CD123 specific chimeric antigen receptors for cancer immunotherapy

ABSTRACT

The present invention relates to Chimeric Antigen Receptors (CAR) that are recombinant chimeric proteins able to redirect immune cell specificity and reactivity toward selected membrane antigens, and more particularly in which extracellular ligand binding is a scFV derived from a CD123 monoclonal antibody, conferring specific immunity against CD123 positive cells. The engineered immune cells endowed with such CARs are particularly suited for treating lymphomas and leukemia.

CROSS REFERENCE TO RELATED APPLICATION

This application is a 35 U.S.C. 371 National Phase of PCT ApplicationNo. PCT/EP2015/055848 filed Mar. 19, 2015, which claims priority toDanish Patent Application No. PA201470137 filed Mar. 19, 2014. Thedisclosure of these prior applications are hereby incorporated in theirentirety by reference.

FIELD OF THE INVENTION

The present invention relates to Chimeric Antigen Receptors (CAR) thatare recombinant chimeric proteins able to redirect immune cellspecificity and reactivity toward selected membrane antigens, and moreparticularly in which extracellular ligand binding is a scFV derivedfrom a CD123 monoclonal antibody, conferring specific immunity againstCD123 positive cells. Interleukin 3 receptor alpha chain (CD123) hasbeen identified as being frequently over-expressed on Leukemia tumorcells, especially in the case of acute myeloid leukemia (AML), comparedto normal hematopoietic stem cells. The engineered immune cells endowedwith the CARs according to the invention show higher efficiency in viewof treating lymphomas and leukemia.

BACKGROUND OF THE INVENTION

Adoptive immunotherapy, which involves the transfer of autologousantigen-specific T cells generated ex vivo, is a promising strategy totreat viral infections and cancer. The T cells used for adoptiveimmunotherapy can be generated either by expansion of antigen-specific Tcells or redirection of T cells through genetic engineering (Park,Rosenberg et al. 2011). Transfer of viral antigen specific T cells is awell-established procedure used for the treatment of transplantassociated viral infections and rare viral-related malignancies.Similarly, isolation and transfer of tumor specific T cells has beenshown to be successful in treating melanoma.

Novel specificities in T cells have been successfully generated throughthe genetic transfer of transgenic T cell receptors or chimeric antigenreceptors (CARs) (Jena, Dotti et al. 2010). CARs are synthetic receptorsconsisting of a targeting moiety that is associated with one or moresignaling domains in a single fusion molecule. In general, the bindingmoiety of a CAR consists of an antigen-binding domain of a single-chainantibody (scFv), comprising the light and variable fragments of amonoclonal antibody joined by a flexible linker. Binding moieties basedon receptor or ligand domains have also been used successfully. Thesignaling domains for first generation CARs are derived from thecytoplasmic region of the CD3zeta or the Fc receptor gamma chains. Firstgeneration CARs have been shown to successfully redirect T cellcytotoxicity, however, they failed to provide prolonged expansion andanti-tumor activity in vivo. Signaling domains from co-stimulatorymolecules including CD28, OX-40 (CD134), and 4-1BB (CD137) have beenadded alone (second generation) or in combination (third generation) toenhance survival and increase proliferation of CAR modified T cells.CARs have successfully allowed T cells to be redirected against antigensexpressed at the surface of tumor cells from various malignanciesincluding lymphomas and solid tumors (Jena, Dotti et al. 2010).

Meanwhile, induction treatments for acute myeloid leukemia (AML) haveremained largely unchanged for nearly 50 years and AML remains a diseaseof poor prognosis. Acute myeloid leukemia (AML) is a diseasecharacterized by the rapid proliferation of immature myeloid cells inthe bone marrow resulting in dysfunctional hematopoiesis. Althoughstandard induction chemotherapy can induce complete remissions, manypatients eventually relapse and succumb to the disease, calling for thedevelopment of novel therapeutics for AML.

Recent advances in the immunophenotyping of AML cells have revealedseveral AML associated cell surface antigens that may act as targets forfuture therapies. The interleukin 3 receptor alpha chain (IL-3Rα;CD123—NCBI reference: NP_001254642) has been identified as a potentialimmunotherapeutic target since it is over-expressed on AML tumor cellscompared to normal hematopoietic stem cells. Additionally, two phase Itrials for CD123-specific therapeutics have been completed with bothdrugs displaying good safety profiles (ClinicalTrials.gov ID:NCT00401739 and NCT00397579). Unfortunately, these CD123 targeting drugshad limited efficacy suggesting that alternative, and more potent andspecific therapies targeting CD123 are required to observe anti-leukemicactivity.

A possibly more potent alternative therapy for the treatment of Leukemiacould be the use of T cells expressing chimeric antigen receptors (CARs)that redirect T cell specificity towards cell surface tumor associatedantigens (TAAs) in an MHC-independent manner. Several groups havedeveloped CARs targeting various antigens for the treatment of B-cellmalignancies. However, CAR engineered T cells for the treatment of AMLremain scarce.

In particular, there is still a need to improve construction of CARsthat show better compatibility with T-cell proliferation, in order toallow the cells expressing such CARs to reach significant clinicaladvantage.

In addition, there is a need to improve CD123 CARs having the capacityto proliferate and target selectively CD123 expressing cells.

Further, the use of such CAR expressing immune T cell targeting CD123 incombination with cytotoxic chemotherapy agents as a treatment usuallyemployed as anti-cancer treatments remains a problem.

Several cytotoxic agents such as anti-metabolites, alkylating agents,anthracyclines, DNA methyltransferase inhibitors, platinum compounds andspindle poisons have been developed to kill cancer cells, in particularcancer cells expressing CD123.

These chemotherapy agents can be detrimental to the establishment ofrobust anti-tumor immunocompetent cells due to their non-specifictoxicity. Small molecule-based therapies targeting cell proliferationpathways may also hamper the establishment of anti-tumor immunity.

Thus, there is also a need of developing T cells targeting CD123 thatwould be specific and compatible with the use of drugs, in particular ofanti-cancer chemotherapies, such as those affecting cell proliferation.

Thus, to use “off-the-shelf” allogeneic therapeutic cells in conjunctionwith chemotherapy, the inventors develop a method of engineeringallogeneic T-cell, less allogenic and resistant to chemotherapeuticagents. The therapeutic benefits afforded by this strategy should beenhanced by the synergistic effects between chemotherapy andimmunotherapy. Moreover, drug resistance can also benefit from theability to selectively expand the engineered T-cell thereby avoiding theproblems due to inefficient gene transfer to these cells.

SUMMARY OF THE INVENTION

The inventors have generated CD123 specific CAR having different designand comprising different scFV derived from CD123 specific antibodies.These CD123 specific CAR are designated CD123 specific CAR or anti-CD123CAR, or 123 CAR, or “CAR of the invention” indiscriminately.

In particular, The Inventors have developed CD123 specific CARcomprising a scFV derived from Klon43 with different architectures andidentified highly specific and very selective CARs constructions thatbind to CD123 expressing cells and selectively destroy CD123 expressingcancer cells.

Following non-specific activation in vitro (e.g. with anti CD3/CD28coated beads and recombinant IL2), T-cells from donors have beentransformed with polynucleotides expressing these CARs using viraltransduction. In certain instances, the T-cells were further engineeredto create non-alloreactive T-cells, more especially by disruption of acomponent of TCR (αβ-T-Cell receptors) to prevent Graft versus hostreaction.

T-cells were further engineered to create T cells resistant toanti-cancer drugs, to be used in combination with said classicalanti-cancer drugs.

The resulting engineered T-cells displayed reactivity in-vitro againstCD123 positive cells to various extend, showing that the CARs of thepresent invention contribute to antigen dependent activation, and alsoproliferation, of the T-cells, making them useful for immunotherapy.

The resulting engineered T-cells displayed reactivity in-vivo againstCD123 positive cells and significantly reduce the number of cancer cellsin vivo.

The engineered T-cells of the invention are designed to display in-vivoreactivity against CD123 positive cells, can be used in concomitancewith anti-cancer drugs, are well tolerated. In a particular embodiment,the engineered T-cells of the invention remain efficient even afterseveral administrations, making them useful for immunotherapy as a firsttreatment (induction), as a consolidation treatment, as a treatment incombination with classical anticancer chemotherapy. The polypeptides andpolynucleotide sequences encoding the CARs of the present invention aredetailed in the present specification.

The engineered immune cells of the present invention are particularlyuseful for therapeutic applications such as B-cell lymphoma or leukemiatreatments.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Schematic representation of an engineered immune cell accordingto the invention. The engineered immune cell presented in this figure isa T-cell transduced with a retroviral polypeptide encoding CAR. ThisT-cell is further engineered to allow a better and safer engraftmentinto the patient, which is optional within the frame of the presentinvention. X gene may be for instance a gene expressing a component ofTCR (TCRalpha or TCRbeta), Y may be a gene involved into the sensitivityof T-cells to immune-suppressive drugs like CD52 (with respect toCampath) or HPRT (with respect to 6-Thioguanine).

FIG. 2: Schematic representation of the different CAR Architecture (V1to V6) of the invention (123 CAR)

FIG. 3: shows the different architectures for the CAR according to theinvention, each of which differs in the hinge region used.

FIG. 4: shows degranulation activity in percentage (%) of degranulationof the 6 different scFv's for one single architecture (v3:CD8-hinge/CD8-transmembrane), when CAR+ T-cells were co-cultured for 6hours with CD123 expressing cells (RPMI8226), or with cells that do notexpress CD123 (K562). White bars correspond to degranulation signalsobserved in T-cells that were cultured alone, black bars represent thesignals observed when T-cells were co-cultured with RPMI8226 cells, andgray bars show degranulation signals of T-cells co-cultured with K562cells.

FIG. 5: shows the degranulation activity (CD107a+ cells) in meanfluorescence intensity (MFI) of CAR T-cells after 6 h co-cultures withCD123neg cells (K562) or cells expressing high or low levels of CD123(RPMI8226 and KG1a, respectively).

FIG. 6: shows the percentage (%) of degranulation, of various anti-CD123CAR T cells when co-cultured for 6 h with cells expressing differentlevels of CD123 (KG1a or RPMI8226), or with cells that do not expressCD123 (K562).

FIG. 7: shows the amount of IFN gamma (IFNγ) released by variousanti-CD123 CAR T cells when co-cultured for 24 h with cells expressingdifferent levels of CD123 (KG1a or RPMI8226), or with cells that do notexpress CD123 (K562).

FIG. 8: shows the specific cytolytic activity of various anti-CD123 CART cells. Assays were done 48 h after CAR mRNA transfection. T-cells wereco-cultured with K562+KG1a or K562+RPMI8226 cells. Cellular viabilityfor each of the cell lines was determined at the end of the co-culturedand a specific cell lysis percentage was calculated.

FIG. 9: shows the general construction used for transduction of T cellsand the percentage (%) of T cells expressing the CAR or BFP at Day 8 or10 post transduction for two different donors analyzed by flowcytometry. The CARs correspond to CAR construction Klon 43-v3 CAR and32716-V3 CAR.

FIG. 10: represents the degranulation activity of T-cells expressingKlon 43-v3 CAR and 32716-V3 CAR, against different cells lines (Daudiand K562 cells that do not express CD123, KG1a, MOLM13 and RPMI8226 thatexpress increasing levels of CD123 (KG1a<MOLM13<RPMI8226). The % ofCD107a+ cells (among CD8+ cells) for three independent donors is givenin the upper panel, and the intensity of CD107a staining is shown in thelower panel for a representative donor. NTD stands for Non Transducedcells.

FIG. 11: shows the IFN gamma release upon 24 h co-culture of Klon 43-v3CAR and 32716-V3 CAR expressing T-cells with different cell lines.

FIG. 12: shows the specific cytolytic activity of Klon 43-v3 CAR and32716-V3 CAR expressing-T cells. A specific cell lysis percentage wascalculated. The results represent results obtained in at least twoindependent donors.

FIG. 13: shows a degranulation activity (in percentage (%) ofdegranulation) of Klon 43-v3 CAR and 32716-V3 CAR expressing-T cells.

FIG. 14: shows a degranulation activity (in MFI) of Klon 43-v3 CAR and32716-V3 CAR expressing-T cells.

FIG. 15: shows the in vivo activity of T-cells expressing the Klon43-v3CAR in NOG Immunodefficient mice. Mice were injected withMOLM13-Luciferase cells either 2 or 7 days before injection ofnon-transduced human T-cells, and with different doses of anti-CD123CAR+ T-cells. The results represent the bioluminescent signal observedat different time points after T-cell injection (mean of 4 mice in eachgroup, except for G12, in which 1 of the 4 mice died between days 21 and28).

TABLE 1 Sequence of the different CAR components Functional domainsSEQ ID # Raw amino acid sequence CD8α signal peptide SEQ ID NO. 1MALPVTALLLPLALLLHAARP Alternative signal peptide SEQ ID NO. 2METDTLLLWVLLLWVPGSTG FcγRIIIα hinge SEQ ID NO. 3 GLAVSTISSFFPPGYQ CD8αhinge SEQ ID NO. 4 TTTPAPRPPTPAPTIASQPLSLRPEACRPAAG GAVHTRGLDFACDIgG1 hinge SEQ ID NO. 5 EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK CD8α transmembrane domain SEQ ID NO. 6IYIWAPLAGTCGVLLLSLVITLYC 41BB transmembrane domain SEQ ID NO. 7IISFFLALTSTALLFLLFFLTLRFSVV 41BB intracellular domain SEQ ID NO. 8KRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCEL CD3ζ intracellular domainSEQ ID NO. 9 RVKFSRSADAPAYQQGQNQLYNELNLGRR EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD GLYQGLSTATKDTYDALHMQALPPR LinkerSEQ ID NO. 10 GGGGSGGGGSGGGGS

TABLE 2 Sequence of the different CAR components ScFv sequences SEQ ID #Raw amino acid sequence 7G3 heavy chain  SEQ ID MGWSWIFLFLVSGTGGVLSEVQLQQSGPELV variable region NO. 11KPGASVKMSCKASGYTFTDYYMKWVKQSH GKSLEWIGDIIPSNGATFYNQKFKGKATLTVDRSSSTAYMHLNSLTSEDSAVYYCTRSHLLRAS WFAYWGQGTLVTVSAAS 7G3 light chain SEQ ID  MESQTQVLMSLLFWVSGTCGDFVMTQSPSSL variable region NO. 12TVTAGEKVTMSCKSSQSLLNSGNQKNYLTW YLQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPYTFG GGTKLEIKR Old4 heavy chain  SEQ ID WTWRFLFVVAAATGVQSQVQLLQSGAEVKK variable region NO. 13PGSSVKVSCKASGGTFSTYAISWVRQAPGQG LEWMGGIIPIFGIVNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGGSGPDVLD IWGQGTMVTVSSAST Old4 light chain SEQ ID  MDMRVPAQLLGLLLLWLPGARCVIWMTQSP variable region NO. 14SLLSASTGDRVTISCRMSQGIRSYLAWYQQKP GKAPELLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQSEDFATYYCQQYYSFPYTFGQGTKLEI KRTV 26292 heavy chain  SEQ ID QVQLQQPGAELVRPGASVKLSCKASGYTFTS variable region NO. 15YWMNWVKQRPDQGLEWIGRIDPYDSETHYN QKFKDKAILTVDKSSSTAYMQLSSLTSEDSAVYYCARGNWDDYWGQGTTLTVSS 26292 light chain  SEQ ID DVQITQSPSYLAASPGETITINCRASKSISKDLA variable region NO. 16WYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGS GTDFTLTISSLEPEDFAMYYCQQHNKYPYTFGGGTKLEIK 32716 heavy chain  SEQ ID  QIQLVQSGPELKKPGETVKISCKASGYIFTNYvariable region NO. 17 GMNWVKQAPGKSFKWMGWINTYTGESTYSADFKGRFAFSLETSASTAYLHINDLKNEDTAT YFCARSGGYDPMDYWGQGTSVTVSS32716 light chain  SEQ ID  DIVLTQSPASLAVSLGQRATISCRASESVDNYvariable region NO. 18 GNTFMHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQS NEDPPTFGAGTKLELK Klon43 heavy chain SEQ ID  EVKLVESGGGLVQPGGSLSLSCAASGFTFTDY variable region NO. 19YMSWVRQPPGKALEWLALIRSKADGYTTEYS ASVKGRFTLSRDDSQSILYLQMNALRPEDSATYYCARDAAYYSYYSPEGAMDYWGQGTSVTVSS Klon43 light chain  SEQ ID MADYKDIVMTQSHKFMSTSVGDRVNITCKAS variable region NO. 20QNVDSAVAWYQQKPGQSPKALIYSASYRYSG VPDRFTGRGSGTDFTLTISSVQAEDLAVYYCQQYYSTPWTFGGGTKLEIKR 12F1 heavy chain  SEQ ID VQLQESGPGLVKPSQSLSLTCSVTDYSITSGY variable region NO. 21YWNWIRQFPGNKLEWMGYISYDGSNNYNPS LKNRISITRDTSKNQFFLKLSSVTTEDTATYYCSRGEGFYFDSWGQGTTLTVSSARS 12F1 light chain  SEQ ID DIMMSQSPSSLAVSVGEKFTMTCKSSQSLFFG variable region NO. 22STQKNYLAWYQQKPGQSPKLLIYWASTRESG VPDRFTGSGSGTDFTLAISSVMPEDLAVYYCQQYYNYPWTFGGGTKLEIK

TABLE 3 CAR of structure V-1 CAR Structure CAR signal Designationpeptide FcγRIIIα V-1 (optional) VH VL hinge CD8α TM 41BB-IC CD3ζ CD7G3-1 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO.11 NO. 12 NO. 3 NO. 6 NO. 8 NO. 9 NO. 23) 26292-1 SEQ ID SEQ ID SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 15 NO. 16 NO. 3 NO. 6 NO.8 NO. 9 NO. 30) 32716-1 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID(SEQ ID NO. 1 NO. 17 NO. 18 NO. 3 NO. 6 NO. 8 NO. 9 NO. 36) Klo43-1 SEQID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO. 1 NO. 19 NO. 20NO. 3 NO. 6 NO. 8 NO. 9 NO. 42)

TABLE 4 CAR of structure V-2 CAR Structure CAR signal Designationpeptide FcγRIIIα V-2 (optional) VH VL hinge 41BB-TM 41BB-IC CD3ζ CD7G3-2 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO.11 NO. 12 NO. 3 NO. 7 NO. 8 NO. 9 NO. 24) 26292-2 SEQ ID SEQ ID SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 15 NO. 16 NO. 3 NO. 7 NO.8 NO. 9 NO. 31) 32716-2 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID(SEQ ID NO. 1 NO. 17 NO. 18 NO. 3 NO. 7 NO. 8 NO. 9 NO. 37) Klo43-2 SEQID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 19 NO. 20NO. 3 NO. 7 NO. 8 NO. 9 NO. 43)

TABLE 5 CAR of structure V-3 CAR Structure CAR signal Designationpeptide CD8α V-3 (optional) VH VL hinge CD8α TM 41BB-IC CD3ζ CD 7G3-3SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 11NO. 12 NO. 4 NO. 6 NO. 8 NO. 9 NO. 25) Old4-3 SEQ ID SEQ ID SEQ ID SEQID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 13 NO. 14 NO. 4 NO. 6 NO. 8NO. 9 NO. 29) 26292-3 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID(SEQ ID NO. 1 NO. 15 NO. 16 NO. 4 NO. 6 NO. 8 NO. 9 NO. 32) 32716-3 SEQID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 17 NO. 18NO. 4 NO. 6 NO. 8 NO. 9 NO. 38) Klo43-3 SEQ ID SEQ ID SEQ ID SEQ ID SEQID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 19 NO. 20 NO. 4 NO. 6 NO. 8 NO. 9 NO.44) 12SF1-3 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO.1 NO. 21 NO. 22 NO. 4 NO. 6 NO. 8 NO. 9 NO. 48)

TABLE 6 CAR of structure V-4 CAR Structure CAR signal Designationpeptide CD8α V-4 (optional) VH VL hinge 41BB-TM 41BB-IC CD3ζ CD 7G3-4SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 11NO. 12 NO. 4 NO. 7 NO. 8 NO. 9 NO. 26) 26292-4 SEQ ID SEQ ID SEQ ID SEQID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 15 NO. 16 NO. 4 NO. 7 NO. 8NO. 9 NO. 33) 32716-4 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID(SEQ ID NO. 1 NO. 17 NO. 18 NO. 4 NO. 7 NO. 8 NO. 9 NO. 39) Klo43-4 SEQID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 19 NO. 20NO. 4 NO. 7 NO. 8 NO. 9 NO. 45)

TABLE 7 CAR of structure V-5 CAR Structure CAR signal Designationpeptide IgG1 V-5 (optional) VH VL hinge CD8α TM 41BB-IC CD3ζ CD 7G3-5SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 11NO. 12 NO. 5 NO. 6 NO. 8 NO. 9 NO. 27) 26292-5 SEQ ID SEQ ID SEQ ID SEQID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 15 NO. 16 NO. 5 NO. 6 NO. 8NO. 9 NO. 34) 32716-5 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID(SEQ ID NO. 1 NO. 17 NO. 18 NO. 5 NO. 6 NO. 8 NO. 9 NO. 40) Klo43-5 SEQID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 19 NO. 20NO. 5 NO. 6 NO. 8 NO. 9 NO. 46)

TABLE 8 CAR of structure V-6 CAR Structure CAR signal Designationpeptide IgG1 V-6 (optional) VH VL hinge 41BB-TM 41BB-IC CD3ζ CD 7G3-6SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 11NO. 12 NO. 5 NO. 7 NO. 8 NO. 9 NO. 28) 26292-6 SEQ ID SEQ ID SEQ ID SEQID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 15 NO. 16 NO. 5 NO. 7 NO. 8NO. 9 NO. 35) 32716-6 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID(SEQ ID NO. 1 NO. 17 NO. 18 NO. 5 NO. 7 NO. 8 NO. 9 NO. 41) Klo43-6 SEQID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 1 NO. 19 NO. 20NO. 5 NO. 7 NO. 8 NO. 9 NO. 47)

DETAILED DESCRIPTION OF THE INVENTION

Unless specifically defined herein, all technical and scientific termsused have the same meaning as commonly understood by a skilled artisanin the fields of gene therapy, biochemistry, genetics, and molecularbiology.

All methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,with suitable methods and materials being described herein. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willprevail. Further, the materials, methods, and examples are illustrativeonly and are not intended to be limiting, unless otherwise specified.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, CurrentProtocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley andson Inc, Library of Congress, USA); Molecular Cloning: A LaboratoryManual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, NewYork: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis(M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; NucleicAcid Hybridization (B. D. Harries & S. J. Higgins eds. 1984);Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984);Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987);Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A PracticalGuide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J.Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York),specifically, Vols. 154 and 155 (Wu et al. eds.) and Vol. 185, “GeneExpression Technology” (D. Goeddel, ed.); Gene Transfer Vectors ForMammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold SpringHarbor Laboratory); Immunochemical Methods In Cell And Molecular Biology(Mayer and Walker, eds., Academic Press, London, 1987); Handbook OfExperimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell,eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1986).

The present invention discloses a CD123 specific chimeric antigenreceptor (“123 CAR” or “CAR”) having one of the polypeptide structureselected from V1 to V6, as illustrated in FIG. 2, said structurecomprising an extra cellular ligand binding-domain comprising VH and VLfrom a monoclonal anti-CD123 antibody, a hinge, a transmembrane domain,a cytoplasmic domain including a CD3 zeta signaling domain and aco-stimulatory domain from 4-1BB, said 123 CAR having at least 80%sequence identity with either SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO.46, SEQ ID NO. 29 or SEQ ID NO. 48.

The present invention discloses a CD123 specific chimeric antigenreceptor (123 CAR) having one of the polypeptide structure selected fromV1, V3 and V5, as illustrated in FIG. 2, said structure comprising anextra cellular ligand binding-domain comprising VH and VL from amonoclonal anti-CD123 antibody, a hinge, a transmembrane domain, acytoplasmic domain including a CD3 zeta signaling domain and aco-stimulatory domain from 4-1BB, said 123 CAR having at least 80%sequence identity with either SEQ ID NO. 42, SEQ ID NO. 44 or SEQ ID NO.46.

The present invention discloses a CD123 specific chimeric antigenreceptor (123 CAR) having one of the polypeptide structure selected fromV1, V3 and V5, as illustrated in FIG. 2, said structure comprising anextra cellular ligand binding-domain comprising VH and VL from amonoclonal anti-CD123 antibody, a hinge, a transmembrane domain, acytoplasmic domain including a CD3 zeta signaling domain and aco-stimulatory domain from 4-1BB, said 123 CAR having at least 80%sequence identity with either SEQ ID NO. 75, SEQ ID NO. 76, SEQ ID NO.77.

The present invention discloses a specific chimeric antigen receptor(123 CAR) having a polypeptide structure V3 as illustrated in FIG. 2,and described above wherein said 123 CAR has at least 80% sequenceidentity with SEQ ID NO. 42.

The present invention discloses a specific chimeric antigen receptor(123 CAR) having a polypeptide structure V3 as illustrated in FIG. 2,and described above, wherein said 123 CAR has at least 80% sequenceidentity with SEQ ID NO. 44.

The present invention discloses a specific chimeric antigen receptor(123 CAR) having a polypeptide structure V3 as illustrated in FIG. 2,and described above wherein said 123 CAR has at least 80% sequenceidentity with SEQ ID NO. 46.

The present invention discloses a specific chimeric antigen receptor(123 CAR) having a polypeptide structure V3 as illustrated in FIG. 2,and described above wherein said 123 CAR has at least 80% sequenceidentity with SEQ ID NO. 75.

The present invention discloses a specific chimeric antigen receptor(123 CAR) having a polypeptide structure V3 as illustrated in FIG. 2,and described above wherein said 123 CAR has at least 80% sequenceidentity with SEQ ID NO. 76.

The present invention discloses a specific chimeric antigen receptor(123 CAR) having a polypeptide structure V3 as illustrated in FIG. 2,and described above wherein said 123 CAR has at least 80% sequenceidentity with SEQ ID NO. 77.

The present invention discloses a CD123 specific chimeric antigenreceptor (CAR) having a polypeptide structure V3 as illustrated in FIG.2, and described above said structure comprising an extra cellularligand binding-domain VH and VL from a monoclonal anti-CD123 antibodycomprising the following CDR sequences:—GFTFTDYY (SEQ ID NO 67),

-   -   RSKADGYTT (SEQ ID NO 68),    -   ARDAAYYSYYSPEGAMDY (SEQ ID NO 69), and    -   QNVDSA (SEQ ID NO 70),    -   SAS (SEQ ID NO 71),    -   QQYYSTPWT (SEQ ID NO 72),        -   and said structure comprising:            a hinge, a transmembrane domain and a cytoplasmic domain            including a CD3 zeta signaling domain and a co-stimulatory            domain from 4-1BB.

The present invention discloses a CD123 specific chimeric antigenreceptor (123 CAR) as above, wherein said extra cellular ligandbinding-domain VH and VL is humanized.

The present invention discloses a CD123 specific chimeric antigenreceptor (123 CAR) as above, wherein said extra cellular ligandbinding-domain is humanized.

The present invention discloses a CD123 specific chimeric antigenreceptor (123 CAR) as above, said CD123 specific chimeric antigenreceptor is humanized.

The present invention discloses a CD123 specific chimeric antigenreceptor (123 CAR) as above, wherein said extra cellular ligandbinding-domain VH and VL from a monoclonal anti-CD123 antibody comprisesthe following sequence

(SEQ ID No 73) XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXGFTFTDYYXXXXXXXXXXXXXXXXXIRSKADGYTTXXXXXXXXXXXXXXXXXXXXXXXXXXXXARDAAYYSYYSPEGAMDYXXXXXXXXXXX and (SEQ ID No 74)XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXQNVDSAXXXXXXXXXXXXXXXXXSASXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXQQYYSTP WTXXXXXXXXX,an amino acid can be anyone of the amino acid, for example alanine,asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycinehistidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine, valine, aspartic acid,glutamic acid.

The present invention discloses a CD123 specific chimeric antigenreceptor (CAR) as described above, wherein said extra cellular ligandbinding-domain VH and VL from a monoclonal anti-CD123 antibodyrespectively comprise at least one of the following sequences:

(Variant VH1: SEQ ID NO. 60):EVKLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (Variant VH2: SEQ ID NO. 61):EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (Variant VH3: SEQ ID NO. 62):EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (Variant VH4: SEQ ID NO. 63):EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (Variant VH5: SEQ ID NO. 64):EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (Variant VH6: SEQ ID NO. 65):EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (Variant VH7: SEQ ID NO. 66):EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRDAAYYSYYSPEGAMDYWGQGTLVTVSS, Variant VL1: SEQ ID NO. 54):MADYKDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR,Variant VL2: SEQ ID NO. 55):MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR,Variant VL3: SEQ ID NO. 56):MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR,Variant VL4: SEQ ID NO. 57):MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR,Variant VL5: SEQ ID NO. 58):MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR, andVariant VL6: SEQ ID NO. 59):MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYGQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR,or a combination thereof.

The present invention discloses a CD123 specific chimeric antigenreceptor (CAR) as described above, wherein said extra cellular ligandbinding-domain VH and VL from a monoclonal anti-CD123 antibodyrespectively comprise at least one of the following sequences:

(SEQ ID NO. 60) EVKLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (SEQ ID NO. 61)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (SEQ ID NO. 62)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (SEQ ID NO. 63)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (SEQ ID NO. 64)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (SEQ ID NO. 65)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (SEQ ID NO. 66)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRDAAYYSYYSPEGAMDYWGQGTLVTVSS,

(SEQ ID NO. 54) MADYKDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR,(SEQ ID NO. 55) MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR,(SEQ ID NO. 56) MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR,(SEQ ID NO. 57) MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR,(SEQ ID NO. 58) MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR,(SEQ ID NO. 59) MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYGQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR,or a combination thereof.

Advantageously, the present invention discloses a CD123 specificchimeric antigen receptor (CAR) as described above, wherein said extracellular ligand binding-domain VH and VL from a monoclonal anti-CD123antibody respectively comprise at least one of the following sequences:

(SEQ ID NO. 60) EVKLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (SEQ ID NO. 61)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (SEQ ID NO. 62)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (SEQ ID NO. 63)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (SEQ ID NO. 64)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (SEQ ID NO. 65)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (SEQ ID NO. 66)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRDAAYYSYYSPEGAMDYWGQGTLVTVSS, and at leat one of the following sequences:(SEQ ID NO. 54) MADYKDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR,(SEQ ID NO. 55) MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR,(SEQ ID NO. 56) MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR,(SEQ ID NO. 57) MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR,(SEQ ID NO. 58) MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR,(SEQ ID NO. 59) MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYGQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR.

The present invention discloses a CD123 specific CAR as described above,wherein said structure V3 comprises a CD8 alpha hinge and a CD8 alphatransmembrane domain.

The present invention discloses a CD123 specific CAR as described above,wherein said structure V3 comprises a CD8 alpha hinge, 41BBB cytoplasmicdomain and a CD8 alpha transmembrane domain.

The present invention discloses a CD123 specific CAR as described above,wherein said structure V3 comprises a CD8 alpha hinge and a 4-1BBtransmembrane domain.

The present invention discloses a CD123 specific CAR as above, whereinsaid VH and VL have at least 80% identity with a polypeptide sequenceselected from SEQ ID NO. 19 and SEQ ID NO. 20.

The present invention discloses a CD123 specific CAR as above, whereinsaid VH and VL have at least 80% identity with a polypeptide of SEQ IDNO. 19 and/or of SEQ ID NO. 20.

The present invention discloses a CD123 specific CAR as above furthercomprising another extracellular ligand binding domain which is notspecific for CD123.

The present invention discloses a CD123 specific CAR as above, furthercomprising a signal peptide, preferably of SEQ ID NO 1 or SEQ ID NO 2.

The present invention discloses a CD123 specific CAR as above, wherein alinker of SEQ ID NO 10 is inserted between VH and VL.

The present invention discloses a polynucleotide encoding a CD123specific chimeric antigen receptor according to any one of the CD123specific CAR described above, said polynucleotide further comprising asignal peptide, preferably of SEQ ID NO 1 or SEQ ID NO 2.

The present invention discloses an expression vector comprising apolynucleotide as above.

The present invention discloses an engineered immune cell expressing atthe cell surface membrane a CD123 specific chimeric antigen receptor asdescribed above, preferably an engineered immune cell that canexpressing at the cell surface membrane a CD123 specific chimericantigen receptor as described above.

The present invention discloses an engineered immune cell as above,derived from T-lymphocytes, optionally resistant to an anti-cancer drug,and bearing a deletion in a gene coding an alpha TCR or a beta TCR.

The present invention discloses an engineered immune cell as above,wherein expression of TCR is suppressed.

The present invention discloses an engineered immune cell as above,wherein expression of at least one MHC protein, preferably β2m or HLA,is suppressed in said engineered immune cell. β2m stands for beta 2microglobulin and HLA for human leukocyte antigen. The MHC protein is aMHC protein of Class I or of class II.

The present invention discloses an engineered immune cell as above,wherein said engineered immune cell is mutated to confer resistance toat least one immune suppressive drug, chemotherapy drug, or anti-cancerdrug.

The present invention discloses an engineered immune cell as above foruse in therapy.

The present invention discloses an engineered immune cell for use intherapy as above, wherein the patient is a human.

The present invention discloses an engineered immune cell for use intherapy as above, wherein the condition is a pre-malignant or malignantcancer condition characterized by CD123-expressing cells.

The present invention discloses an engineered immune cell for use intherapy as above, wherein the condition is a condition which ischaracterized by an overabundance of CD123-expressing cells.

The present invention discloses an engineered immune cell for use intherapy as above, wherein the malignant cancer condition is ahaematological cancer condition.

The present invention discloses an engineered immune cell for use intherapy as above, wherein the haematological cancer condition isleukemia or malignant lymphoproliferative disorders.

The present invention discloses an engineered immune cell for use intherapy as above, wherein said leukemia is selected from the groupconsisting of acute myelogenous leukemia, chronic myelogenous leukemia,myelodysplastic syndrome, acute lymphoid leukemia, chronic lymphoidleukemia, and myelodysplastic syndrome.

The present invention discloses an engineered immune cell for use intherapy as above, wherein the leukemia is acute myelogenous leukemia(AML).

The present invention discloses an engineered immune cell for use intherapy as above, wherein said hematologic cancer is a malignantlymphoproliferative disorder.

The present invention discloses an engineered immune cell for use intherapy as above, wherein said malignant lymphoproliferative disorder islymphoma.

The present invention discloses an engineered immune cell for use intherapy as above, wherein said lymphoma is selected from the groupconsisting of multiple myeloma, non-Hodgkin's lymphoma, Burkitt'slymphoma, and follicular lymphoma (small cell and large cell).

The present invention discloses a method of impairing a hematologiccancer cell comprising contacting said hematologic cancer cell with anengineered cell according to any one of claims 13 to 17 in an amounteffective to cause impairment of said cancer cell.

The present invention discloses a method of engineering an immune cellcomprising:

-   -   (a) Providing an immune cell,    -   (b) Expressing at the surface of said cell at least one CD123        specific chimeric antigen receptor according to any one of        claims 1 to 10.    -   The present invention discloses a method of engineering an        immune cell as above comprising:    -   (a) Providing an immune cell,    -   (b) Introducing into said cell at least one polynucleotide        encoding said CD123 specific chimeric antigen receptor,    -   (c) Expressing said polynucleotide into said cell.

The present invention discloses a method of engineering an immune cellas above comprising:

-   -   (a) Providing an immune cell,    -   (b) Introducing into said cell at least one polynucleotide        encoding said CD123 specific chimeric antigen receptor,    -   (c) Introducing at least one other chimeric antigen receptor        which is not specific for CD123.

The present invention discloses a method of treating a subject in needthereof comprising:

-   -   (a) Providing an immune cell expressing at the surface a CD123        specific Chimeric Antigen Receptor according to any one of        claims 1 to 10    -   (b) Administrating said immune cells to said patient.    -   The present invention discloses a method of treating a subject        in need thereof as above, wherein said immune cell is provided        from a donor.    -   The present invention discloses a method of treating a subject        in need thereof as above, wherein said immune cell is provided        from the patient himself.        CD123 specific Chimeric Antigen Receptors

The present invention relates to new designs of anti-CD123 chimericantigen receptor (CAR) comprising an extracellular ligand-bindingdomain, a transmembrane domain and a signaling transducing domain.

The term “extracellular ligand-binding domain” as used herein is definedas an oligo- or polypeptide that is capable of binding a ligand.Preferably, the domain will be capable of interacting with a cellsurface molecule. For example, the extracellular ligand-binding domainmay be chosen to recognize a ligand that acts as a cell surface markeron target cells associated with a particular disease state. In apreferred embodiment, said extracellular ligand-binding domain comprisesa single chain antibody fragment (scFv) comprising the light (V_(L)) andthe heavy (V_(H)) variable fragment of a target antigen specificmonoclonal anti CD-123 antibody joined by a flexible linker. Said V_(L)and V_(H) are preferably selected from the antibodies referred to in theliterature as 7G3, Old4, 26292, 32716, Klon43 and 12F1 as indicated inTable 1 to 8, more preferably Old4, Klon43 and 12F1, and even morepreferably, Klon43. They are preferably linked together by a flexiblelinker comprising the sequence SEQ ID NO. 10. In other words, said CARspreferentially comprise an extracellular ligand-biding domain comprisinga polypeptide sequence displaying at least 90%, 95% 97% or 99% identitywith an amino acid sequence selected from the group consisting of SEQ IDNO: 11 to SEQ ID NO: 22 (see Table 2).

More preferably, said CARs preferentially comprise an extracellularligand-biding domain comprising a polypeptide sequence displaying atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acidsequence selected from the group consisting of SEQ ID NO: 13, 14, 19,20, 21 to SEQ ID NO: 22 and even more preferably said CARspreferentially comprise an extracellular ligand-biding domain comprisinga polypeptide sequence displaying at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity with amino acid sequences consisting of SEQ ID NO:19 and/or 20.

Even more preferably, said CARs comprise an extracellular ligand-bidingdomain comprising a polypeptide sequence displaying at least 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with an amino acid sequence selected fromthe group consisting of SEQ ID NO: 1+SEQ ID NO: 13, SEQ ID NO: 1+SEQ IDNO: 14, SEQ ID NO: 1+SEQ ID NO: 19, SEQ ID NO: 1+SEQ ID NO: 20, SEQ IDNO: 1+SEQ ID NO: 21 and SEQ ID NO: 1+SEQ ID NO: 22 and even morepreferably said CARs preferentially comprise an extracellularligand-biding domain comprising a polypeptide sequence displaying atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with amino acid sequencesconsisting of SEQ ID NO: 1+SEQ ID NO:19 and SEQ ID NO: 1+SEQ ID NO: 20.

By the term “recombinant antibody” as used herein, is meant an antibodyor antibody fragment which is generated using recombinant DNAtechnology, such as, for example, an antibody or antibody fragmentexpressed by a bacteriophage, a yeast expression system or a mammaliancell expression system. The term should also be construed to mean anantibody or antibody fragment which has been generated by the synthesisof a DNA molecule encoding the antibody or antibody fragment and whichDNA molecule expresses an antibody or antibody fragment protein, or anamino acid sequence specifying the antibody or antibody fragment,wherein the DNA or amino acid sequence has been obtained usingrecombinant or synthetic DNA or amino acid sequence technology which isavailable and well known in the art.

As used herein, the term “conservative sequence modifications” or“humanization” is intended to refer to amino acid modifications that donot significantly affect or alter the binding characteristics of the CARand/or that do not significantly affect the activity of the CARcontaining the modified amino acid sequence and reduce or abolish ahuman antimouse antibody (HAMA) response. Such conservativemodifications include amino acid substitutions, additions and deletionsin said antibody fragment in said CAR and/or any of the other parts ofsaid CAR molecule. Modifications can be introduced into an antibody,into an antibody fragment or in any of the other parts of the CARmolecule of the invention by standard techniques known in the art, suchas site-directed mutagenesis, PCR-mediated mutagenesis or by employingoptimized germline sequences.

Conservative amino acid substitutions are ones in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine,tryptophan), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one ormore amino acid residues within a CAR of the invention can be replacedwith other amino acid residues from the same side chain family and thealtered CAR can be tested for the ability to bind CD 123 using thefunctional assays described herein.

The signal transducing domain or intracellular signaling domain of a CARaccording to the present invention is responsible for intracellularsignaling following the binding of extracellular ligand binding domainto the target resulting in the activation of the immune cell and immuneresponse. In other words, the signal transducing domain is responsiblefor the activation of at least one of the normal effector functions ofthe immune cell in which the CAR is expressed. For example, the effectorfunction of a T cell can be a cytolytic activity or helper activityincluding the secretion of cytokines. Thus, the term “signal transducingdomain” refers to the portion of a protein which transduces the effectorsignal function signal and directs the cell to perform a specializedfunction.

Preferred examples of signal transducing domain for use in a CAR can bethe cytoplasmic sequences of the T cell receptor and co-receptors thatact in concert to initiate signal transduction following antigenreceptor engagement, as well as any derivate or variant of thesesequences and any synthetic sequence that has the same functionalcapability. Signal transduction domain comprises two distinct classes ofcytoplasmic signaling sequence, those that initiate antigen-dependentprimary activation, and those that act in an antigen-independent mannerto provide a secondary or co-stimulatory signal. Primary cytoplasmicsignaling sequence can comprise signaling motifs which are known asimmunoreceptor tyrosine-based activation motifs of ITAMs. ITAMs are welldefined signaling motifs found in the intracytoplasmic tail of a varietyof receptors that serve as binding sites for syk/zap70 class tyrosinekinases. Examples of ITAM used in the invention can include as nonlimiting examples those derived from TCRzeta, FcRgamma, FcRbeta,FcRepsilon, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b andCD66d. In a preferred embodiment, the signaling transducing domain ofthe CAR can comprise the CD3zeta signaling domain which has amino acidsequence with at least 70%, preferably at least 80%, more preferably atleast 90%, 95% 97% or 99% or 100% sequence identity with amino acidsequence selected from the group consisting of SEQ ID NO: 9.

In particular embodiment the signal transduction domain of the CAR ofthe present invention comprises a co-stimulatory signal molecule. Aco-stimulatory molecule is a cell surface molecule other than an antigenreceptor or their ligands that is required for an efficient immuneresponse. “Co-stimulatory ligand” refers to a molecule on an antigenpresenting cell that specifically binds a cognate co-stimulatorymolecule on a T-cell, thereby providing a signal which, in addition tothe primary signal provided by, for instance, binding of a TCR/CD3complex with an MHC molecule loaded with peptide, mediates a T cellresponse, including, but not limited to, proliferation activation,differentiation and the like. A co-stimulatory ligand can include but isnot limited to CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL,OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesionmolecule (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM,lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, an agonist or antibodythat binds Toll ligand receptor and a ligand that specifically bindswith B7-H3. A co-stimulatory ligand also encompasses, inter alia, anantibody that specifically binds with a co-stimulatory molecule presenton a T cell, such as but not limited to, CD27, CD28, 4-1BB, OX40, CD30,CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2,CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83. A“co-stimulatory molecule” refers to the cognate binding partner on aT-cell that specifically binds with a co-stimulatory ligand, therebymediating a co-stimulatory response by the cell, such as, but notlimited to proliferation. Co-stimulatory molecules include, but are notlimited to an MHC class I molecule, BTLA and Toll ligand receptor.Examples of costimulatory molecules include CD27, CD28, CD8, 4-1BB(CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associatedantigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and a ligand thatspecifically binds with CD83 and the like.

In a preferred embodiment, the signal transduction domain of the CAR ofthe present invention comprises a part of co-stimulatory signal moleculeselected from the group consisting of fragment of 4-1BB (GenBank:AAA53133.) and CD28 (NP_006130.1). In particular the signal transductiondomain of the CAR of the present invention comprises amino acid sequencewhich comprises at least 70%, preferably at least 80%, more preferablyat least 90%, 95% 97% or 99% sequence identity with amino acid sequenceselected from the group consisting of SEQ ID NO: 8.

A CAR according to the present invention is expressed on the surfacemembrane of the cell. Thus, such CAR further comprises a transmembranedomain. The distinguishing features of appropriate transmembrane domainscomprise the ability to be expressed at the surface of a cell,preferably in the present invention an immune cell, in particularlymphocyte cells or Natural killer (NK) cells, and to interact togetherfor directing cellular response of immune cell against a predefinedtarget cell. The transmembrane domain can be derived either from anatural or from a synthetic source. The transmembrane domain can bederived from any membrane-bound or transmembrane protein. Asnon-limiting examples, the transmembrane polypeptide can be a subunit ofthe T-cell receptor such as α, β, γ or

, polypeptide constituting CD3 complex, IL2 receptor p55 (α chain), p75(β chain) or γ chain, subunit chain of Fc receptors, in particular Fcγreceptor III or CD proteins. Alternatively the transmembrane domain canbe synthetic and can comprise predominantly hydrophobic residues such asleucine and valine. In a preferred embodiment said transmembrane domainis derived from the human CD8 alpha chain (e.g. NP_001139345.1) Thetransmembrane domain can further comprise a hinge region between saidextracellular ligand-binding domain and said transmembrane domain. Theterm “hinge region” used herein generally means any oligo- orpolypeptide that functions to link the transmembrane domain to theextracellular ligand-binding domain. In particular, hinge region areused to provide more flexibility and accessibility for the extracellularligand-binding domain. A hinge region may comprise up to 300 aminoacids, preferably 10 to 100 amino acids and most preferably 25 to 50amino acids. Hinge region may be derived from all or part of naturallyoccurring molecules, such as from all or part of the extracellularregion of CD8, CD4 or CD28, or from all or part of an antibody constantregion. Alternatively, the hinge region may be a synthetic sequence thatcorresponds to a naturally occurring hinge sequence, or may be anentirely synthetic hinge sequence. In a preferred embodiment said hingedomain comprises a part of human CD8 alpha chain, FcγRIIIα receptor orIgG1 respectively referred to in this specification as SEQ ID NO. 3, SEQID NO. 4 and SEQ ID NO.5, or hinge polypeptides which display preferablyat least 80%, more preferably at least 90%, 95% 97% or 99% sequenceidentity with these polypeptides.

A car according to the invention generally further comprises atransmembrane domain (TM) more particularly selected from CD8α and4-1BB, showing identity with the polypeptides of SEQ ID NO. 6 or 7,

A CAR according to the invention generally further comprises atransmembrane domain (TM) more particularly a TM selected from CD8α and4-1BB, and even more particularly showing identity with the polypeptidesof SEQ ID NO. 6 or 7,

In a preferred embodiment, a CAR according to the invention furthercomprises a TM domain from CD8α with SEQ ID NO. 6 or showing at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identitywith SEQ ID NO. 6

Downregulation or mutation of target antigens is commonly observed incancer cells, creating antigen-loss escape variants. Thus, to offsettumor escape and render immune cell more specific to target, the CD123specific CAR according to the invention can comprise anotherextracellular ligand-binding domains, to simultaneously bind differentelements in target thereby augmenting immune cell activation andfunction. In one embodiment, the extracellular ligand-binding domainscan be placed in tandem on the same transmembrane polypeptide, andoptionally can be separated by a linker. In another embodiment, saiddifferent extracellular ligand-binding domains can be placed ondifferent transmembrane polypeptides composing the CAR. In anotherembodiment, the present invention relates to a population of CARscomprising each one different extracellular ligand binding domains. In aparticular, the present invention relates to a method of engineeringimmune cells comprising providing an immune cell and expressing at thesurface of said cell a population of CAR each one comprising differentextracellular ligand binding domains. In another particular embodiment,the present invention relates to a method of engineering an immune cellcomprising providing an immune cell and introducing into said cellpolynucleotides encoding polypeptides composing a population of CAR eachone comprising different extracellular ligand binding domains. Bypopulation of CARs, it is meant at least two, three, four, five, six ormore CARs each one comprising different extracellular ligand bindingdomains. The different extracellular ligand binding domains according tothe present invention can preferably simultaneously bind differentelements in target thereby augmenting immune cell activation andfunction. The present invention also relates to an isolated immune cellwhich comprises a population of CARs each one comprising differentextracellular ligand binding domains.

In a preferred embodiment, a CAR according to the invention comprises apolypeptide of SEQ ID NO. 19 and/or a polypeptide of SEQ ID NO. 20, morepreferably a CAR according to the invention comprises a polypeptide withat least 80% identity, preferably 80% to 99% identity with SEQ ID NO. 19and/or a polypeptide having 80 to 99% identity with SEQ ID NO. 20. Evenmore preferably a CAR according to the invention comprises a polypeptidehaving 85 to 99% identity with a polypeptide of SEQ ID NO. 19 and/or SEQID NO. 20.

In a more preferred embodiment, a CAR according to the inventioncomprises a polypeptide having the following sequences:

(SEQ ID NO. 19) EVKLVESGGGLVQPGGSLSLSCAASGFTFTDYYMSWVRQPPGKALEWLALIRSKADGYTTEYSASVKGRFTLSRDDSQSILYLQMNALRPEDSATYYCARDAAYYSYYSPEGAMDYWGQGTSVTVSS and (SEQ ID NO. 20)MADYKDIVMTQSHKFMSTSVGDRVNITCKASQNVDSAVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGRGSGTDFTLTISSVQAEDLAVYYCQQYYSTP WTFGGGTKLEIKR.

In one preferred embodiment, a CAR according to the invention comprisesat least a polypeptide having the following sequence:

(SEQ ID NO. 60) EVKLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS,

In one preferred embodiment, a CAR according to the invention comprisesa polypeptide having the following sequence:

(SEQ ID NO. 61) EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS

In one preferred embodiment, a CAR according to the invention comprisesa polypeptide having the following sequence:

(SEQ ID NO. 62) EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS

In one preferred embodiment, a CAR according to the invention comprisesa polypeptide having the following sequence:

(SEQ ID NO. 63) EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS

In one preferred embodiment, a CAR according to the invention comprisesa polypeptide having the following sequence:

(SEQ ID NO. 64) EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS

In one preferred embodiment, a CAR according to the invention comprisesa polypeptide having the following sequence:

(SEQ ID NO. 65) EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS

In one preferred embodiment, a CAR according to the invention comprisesa polypeptide having the following sequence:

(SEQ ID NO. 66) EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRDAAYYSYYSPEGAMDYWGQGTLVTVSS.

In one preferred embodiment, a CAR according to the invention comprisesa polypeptide having the following sequence:

(SEQ ID NO. 54) MADYKDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR

In one preferred embodiment, a CAR according to the invention comprisesa polypeptide having the following sequence:

(SEQ ID NO. 55) MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR

In one preferred embodiment, a CAR according to the invention comprisesa polypeptide having the following sequence:

(SEQ ID NO. 56) MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR

In one preferred embodiment, a CAR according to the invention comprisesa polypeptide having the following sequence:

(SEQ ID NO. 57) MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR,

In one preferred embodiment, a CAR according to the invention comprisesa polypeptide having the following sequence:

(SEQ ID NO. 58) MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR,

In one preferred embodiment, a CAR according to the invention comprisesa polypeptide having the following sequence:

(SEQ ID NO. 59) MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYGQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR

In one preferred embodiment, a CAR according to the invention comprisesat least one polypeptide selected from the following sequences:

(SEQ ID NO. 60) EVKLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (SEQ ID NO. 61)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (SEQ ID NO. 62)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (SEQ ID NO. 63) EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (SEQ ID NO. 64)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (SEQ ID NO. 65)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS and (SEQ ID NO. 66)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRDAAYYSYYSPEGAMDYWGQGTLVTVSS and at least one sequence selected from the following sequences

(SEQ ID NO. 54) MADYKDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR,(SEQ ID NO. 55) MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR,(SEQ ID NO. 56) MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR,(SEQ ID NO. 57) MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR,(SEQ ID NO. 58) MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR  and(SEQ ID NO. 59) MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYGQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR.

In one preferred embodiment, a CAR according to the invention comprisesone polypeptide selected from the following sequences: SEQ ID NO.54, SEQID NO.55, SEQ ID NO.56, SEQ ID NO.57, SEQ ID NO.58, SEQ ID NO.59, SEQ IDNO.60, SEQ ID NO.61, SEQ ID NO.62, SEQ ID NO.63, SEQ ID NO.64, SEQ IDNO.65, and, SEQ ID NO.66.

In a more preferred embodiment, a CAR according to the inventioncomprises one polypeptide selected from the following sequences: SEQ IDNO.54, SEQ ID NO.55, SEQ ID NO.56, SEQ ID NO.57, SEQ ID NO.58, SEQ IDNO.59, and a peptide selected from the following sequences: SEQ IDNO.60, SEQ ID NO.61, SEQ ID NO.62, SEQ ID NO.63, SEQ ID NO.64, SEQ IDNO.65, and SEQ ID NO.66.

In one embodiment, a CAR according to the invention comprises apolypeptide selected from the list consisting in SEQ ID NO.42, SEQ IDNO.44, and SEQ ID NO.46, preferably a CAR comprising 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identity with a polypeptide of SEQ ID NO.42, a CARcomprising 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a polypeptide ofSEQ ID NO.44, and a CAR comprising 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity with a polypeptide of SEQ ID NO.46.

In a more preferred embodiment, a CAR according to the inventioncomprises a polypeptide comprising 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity with SEQ ID NO. 1+SEQ ID NO. 42.

In another preferred embodiment, a CAR according to the inventioncomprises a polypeptide of SEQ ID NO. 10+SEQ ID NO. 42, even morepreferably a CAR comprising 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity withSEQ ID NO. 10+SEQ ID NO. 42, in particular a CAR comprising 85% to 99%identity with SEQ ID NO. 10+SEQ ID NO. 42.

In a more preferred embodiment, a CAR according to the inventioncomprises a polypeptide comprising 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity with SEQ ID NO. 1+SEQ ID NO. 44.

In an even more preferred embodiment, a CAR according to the inventioncomprises a polypeptide of SEQ ID NO. 10+SEQ ID NO. 44, even morepreferably a CAR comprising 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity withSEQ ID NO. 10+SEQ ID NO. 44, in particular a CAR comprising 85% to 99%identity with SEQ ID NO. 10+SEQ ID NO. 44.

In a more preferred embodiment, a CAR according to the inventioncomprises a polypeptide comprising 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identity with SEQ ID NO. 1+SEQ ID NO. 46.

In an even more preferred embodiment, a CAR according to the inventioncomprises a polypeptide of SEQ ID NO. 10+SEQ ID NO. 46, even morepreferably a CAR comprising 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity withSEQ ID NO. 10+SEQ ID NO. 46, in particular a CAR comprising 85% to 99%identity with SEQ ID NO. 10+SEQ ID NO. 46.

In another preferred embodiment, a CAR according to the inventionconsists in a polypeptide of SEQ ID NO. 1+SEQ ID NO.10+SEQ ID NO. 42even more preferably a CAR is consisting in 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity with SEQ ID NO. SEQ ID NO. 1+SEQ ID NO.10+SEQ ID NO. 42.

In another preferred embodiment, a CAR according to the inventionconsists in a polypeptide of SEQ ID NO. SEQ ID NO. 1+SEQ ID NO.10+SEQ IDNO. 44 even more preferably a CAR is consisting in 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identity with SEQ ID NO. SEQ ID NO. 1+SEQ ID NO.10+SEQ IDNO. 44.

In another preferred embodiment, a CAR according to the inventionconsists in a polypeptide of SEQ ID NO. SEQ ID NO. 1+SEQ ID NO.10+SEQ IDNO. 46 even more preferably a CAR is consisting in 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identity with SEQ ID NO. SEQ ID NO. 1+SEQ ID NO.10+SEQ IDNO. 46.

According to the invention, the immune cells expressing the anti-CD123CAR of the invention trigger an anti-cancer immune response. In apreferred embodiment, the immune cells expressing the CAR of theinvention endowed with the anti-CD123 CAR of the invention does triggeran immune response which does not comprise a human anti-mouse antibody(HAMA) response.

According to the invention, an efficient amount of the engineered immunecell can be administered to a patient in need thereof at least once,twice, or several times, alone or in combination with another treatment.

The present invention concerns a CD123 specific chimeric antigenreceptor (CAR) having one of the polypeptide structure selected from V1to V6 as illustrated in FIG. 2, said structure comprising an extracellular ligand binding-domain comprising VH and VL from a monoclonalanti-CD123 antibody, a hinge, a transmembrane domain and a cytoplasmicdomain including a CD3 zeta signaling domain and a co-stimulatory domainfrom 4-1BB.

Polynucleotides, Vectors:

The present invention also relates to polynucleotides, vectors encodingthe above described CAR according to the invention.

The polynucleotide may consist in an expression cassette or expressionvector (e.g. a plasmid for introduction into a bacterial host cell, or aviral vector such as a baculovirus vector for transfection of an insecthost cell, or a plasmid or viral vector such as a lentivirus fortransfection of a mammalian host cell).

In a particular embodiment, the different nucleic acid sequences can beincluded in one polynucleotide or vector which comprises a nucleic acidsequence encoding ribosomal skip sequence such as a sequence encoding a2A peptide. 2A peptides, which were identified in the Aphthovirussubgroup of picornaviruses, causes a ribosomal “skip” from one codon tothe next without the formation of a peptide bond between the two aminoacids encoded by the codons (see (Donnelly and Elliott 2001; Atkins,Wills et al. 2007; Doronina, Wu et al. 2008)). By “codon” is meant threenucleotides on an mRNA (or on the sense strand of a DNA molecule) thatare translated by a ribosome into one amino acid residue. Thus, twopolypeptides can be synthesized from a single, contiguous open readingframe within an mRNA when the polypeptides are separated by a 2Aoligopeptide sequence that is in frame. Such ribosomal skip mechanismsare well known in the art and are known to be used by several vectorsfor the expression of several proteins encoded by a single messengerRNA.

To direct transmembrane polypeptide into the secretory pathway of a hostcell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in polynucleotide sequenceor vector sequence. The secretory signal sequence is operably linked tothe transmembrane nucleic acid sequence, i.e., the two sequences arejoined in the correct reading frame and positioned to direct the newlysynthesized polypeptide into the secretory pathway of the host cell.Secretory signal sequences are commonly positioned 5′ to the nucleicacid sequence encoding the polypeptide of interest, although certainsecretory signal sequences may be positioned elsewhere in the nucleicacid sequence of interest (see, e.g., Welch et al., U.S. Pat. No.5,037,743; Holland et al., U.S. Pat. No. 5,143,830). In a preferredembodiment the signal peptide comprises the amino acid sequence SEQ IDNO: 1 and 2 or at least 90%, 95% 97% or 99% sequence identity with SEQID NO: 1 and/or 2.

Those skilled in the art will recognize that, in view of the degeneracyof the genetic code, considerable sequence variation is possible amongthese polynucleotide molecules. Preferably, the nucleic acid sequencesof the present invention are codon-optimized for expression in mammaliancells, preferably for expression in human cells. Codon-optimizationrefers to the exchange in a sequence of interest of codons that aregenerally rare in highly expressed genes of a given species by codonsthat are generally frequent in highly expressed genes of such species,such codons encoding the amino acids as the codons that are beingexchanged.

Cells

Cell according to the present invention refers to a cell ofhematopoietic origin functionally involved in the initiation and/orexecution of innate and/or adaptative immune response. Cell according tothe present invention is preferably a T-cell obtained from a donor. SaidT cell according to the present invention can be derived from a stemcell. The stem cells can be adult stem cells, embryonic stem cells, moreparticularly non-human stem cells, cord blood stem cells, progenitorcells, bone marrow stem cells, totipotent stem cells or hematopoieticstem cells. In a preferred embodiment, cells are human cells, inparticular human stem cells.

Representative human stem cells are CD34+ cells. Said isolated cell canalso be a dendritic cell, killer dendritic cell, a mast cell, a NK-cell,a B-cell or a T-cell selected from the group consisting of inflammatoryT-lymphocytes, cytotoxic T-lymphocytes, regulatory T-lymphocytes orhelper T-lymphocytes. In another embodiment, said cell can be derivedfrom the group consisting of CD4+ T-lymphocytes and CD8+ T-lymphocytes.Prior to expansion and genetic modification of the cells of theinvention, a source of cells can be obtained from a subject through avariety of non-limiting methods. Cells can be obtained from a number ofnon-limiting sources, including peripheral blood mononuclear cells, bonemarrow, lymph node tissue, cord blood, thymus tissue, tissue from a siteof infection, ascites, pleural effusion, spleen tissue, and tumors. Incertain embodiments of the present invention, any number of T-cell linesavailable and known to those skilled in the art, may be used. In anotherembodiment, said cell is preferably derived from a healthy donor. Inanother embodiment, said cell is part of a mixed population of cellswhich present different phenotypic characteristics.

Preferably, isolation and preparation of stem cells does not require thedestruction of at least one human embryo. The immune cells can originatefrom the patient, in view of operating autologous treatments, or fromdonors in view of producing allogeneic cells, which can be used inallogeneic treatments.

More preferably the immune cell of the invention express an anti-CD123CAR corresponding to SEQ ID NO 42, SEQ ID NO 44, or SEQ ID NO 46, evenmore preferably the immune cell of the invention express an humanizedanti-CD123 CAR corresponding to humanized SEQ ID NO 42, SEQ ID NO 44, orSEQ ID NO 46.

Methods of Engineering Immune Cells Endowed with CARs:

The present invention encompasses the method of preparing immune cellsfor immunotherapy comprising introducing ex-vivo into said immune cellsthe polynucleotides or vectors encoding the CD123 CAR previouslydescribed in WO2014/130635, WO2013176916, WO2013176915 and incorporatedherein by reference.

In a preferred embodiment, said polynucleotides are included inlentiviral vectors in view of being stably expressed in the immunecells.

According to further embodiments, said method further comprises the stepof genetically modifying said cell to make them more suitable forallogeneic transplantation.

Modifying T-Cell by Inactivating at Least One Gene Encoding a T-CellReceptor (TCR) Component.

According to a first aspect, the immune cell can be made lessallogeneic, for instance, by inactivating at least one gene expressingone or more component of T-cell receptor (TCR) as described in WO2013/176915, which can be combined with the inactivation of a geneencoding or regulating HLA or β2m protein expression. Accordingly therisk of graft versus host syndrome and graft rejection is significantlyreduced.

Accordingly, when the immune cells are T-cells, the present inventionalso provides methods to engineer T-cells that are less allogeneic.

Methods of making cells less allogenic can comprise the step ofinactivating at least one gene encoding a T-Cell Receptor (TCR)component, in particular TCRalpha, TCRbeta genes.

Methods disclosed in WO2013/176915 to prepare CAR expressing immune cellsuitable for allogeneic transplantation, by inactivating one or morecomponent of T-cell receptor (TCR), are all incorporated herein byreference.

The present invention encompasses an anti-CD123 CAR expressing immunecell wherein at least one gene expressing one or more component ofT-cell receptor (TCR) has been inactivated. Thus, the present inventionprovides an anti-CD123 CAR expressing T cell wherein the CAR is derivedfrom Klon 43, in particular having at least 80% identity with SEQ ID N°44 and wherein at least one gene expressing one or more component ofT-cell receptor (TCR) is inactivated.

According to the invention, anti-CD123 CAR immune cells with one or morecomponent of T-cell receptor (TCR) inactivated, are intended to be usedas a medicament.

By inactivating a TCR gene it is intended that the gene of interest isnot expressed in a functional protein form. In particular embodiments,the genetic modification of the method relies on the expression, inprovided cells to engineer, of one rare-cutting endonuclease such thatsaid rare-cutting endonuclease specifically catalyzes cleavage in onetargeted gene thereby inactivating said targeted gene. The nucleic acidstrand breaks caused by the rare-cutting endonuclease are commonlyrepaired through the distinct mechanisms of homologous recombination ornon-homologous end joining (NHEJ). However, NHEJ is an imperfect repairprocess that often results in changes to the DNA sequence at the site ofthe cleavage. Mechanisms involve rejoining of what remains of the twoDNA ends through direct re-ligation (Critchlow and Jackson 1998) or viathe so-called microhomology-mediated end joining (Betts, Brenchley etal. 2003; Ma, Kim et al. 2003). Repair via non-homologous end joining(NHEJ) often results in small insertions or deletions and can be usedfor the creation of specific gene knockouts. Said modification may be asubstitution, deletion, or addition of at least one nucleotide. Cells inwhich a cleavage-induced mutagenesis event, i.e. a mutagenesis eventconsecutive to an NHEJ event, has occurred can be identified and/orselected by well-known method in the art. In a particular embodiment,the step of inactivating at least a gene encoding a component of theT-cell receptor (TCR) into the cells of each individual sample comprisesintroducing into the cell a rare-cutting endonuclease able to disrupt atleast one gene encoding a component of the T-cell receptor (TCR). In amore particular embodiment, said cells of each individual sample aretransformed with nucleic acid encoding a rare-cutting endonucleasecapable of disrupting at least one gene encoding a component of theT-cell receptor (TCR), and said rare-cutting endonuclease is expressedinto said cells.

Said rare-cutting endonuclease can be a meganuclease, a Zinc fingernuclease, CRISPR/Cas9 nuclease, Argonaute nuclease, a TALE-nuclease or aMBBBD-nuclease. In a preferred embodiment, said rare-cuttingendonuclease is a TALE-nuclease. By TALE-nuclease is intended a fusionprotein consisting of a DNA-binding domain derived from a TranscriptionActivator Like Effector (TALE) and one nuclease catalytic domain tocleave a nucleic acid target sequence (Boch, Scholze et al. 2009; Moscouand Bogdanove 2009; Christian, Cermak et al. 2010; Cermak, Doyle et al.2011; Geissler, Scholze et al. 2011; Huang, Xiao et al. 2011; Li, Huanget al. 2011; Mahfouz, Li et al. 2011; Miller, Tan et al. 2011;Morbitzer, Romer et al. 2011; Mussolino, Morbitzer et al. 2011; Sander,Cade et al. 2011; Tesson, Usal et al. 2011; Weber, Gruetzner et al.2011; Zhang, Cong et al. 2011; Deng, Yan et al. 2012; Li, Piatek et al.2012; Mahfouz, Li et al. 2012; Mak, Bradley et al. 2012). In the presentinvention new TALE-nucleases have been designed for precisely targetingrelevant genes for adoptive immunotherapy strategies.

Preferred TALE-nucleases recognizing and cleaving the target sequenceare described in PCT/EP2014/075317. In particular, additional catalyticdomain can be further introduced into the cell with said rare-cuttingendonuclease to increase mutagenesis in order to enhance their capacityto inactivate targeted genes. More particularly, said additionalcatalytic domain is a DNA end processing enzyme. Non limiting examplesof DNA end-processing enzymes include 5-3′ exonucleases, 3-5′exonucleases, 5-3′ alkaline exonucleases, 5′ flap endonucleases,helicases, hosphatase, hydrolases and template-independent DNApolymerases. Non limiting examples of such catalytic domain comprise ofa protein domain or catalytically active derivate of the protein domainselected from the group consisting of hExol (EXO1_HUMAN), Yeast Exol(EXO1_YEAST), E. coli Exol, Human TREX2, Mouse TREX1, Human TREX1,Bovine TREX1, Rat TREX1, TdT (terminal deoxynucleotidyl transferase)Human DNA2, Yeast DNA2 (DNA2_YEAST). In a preferred embodiment, saidadditional catalytic domain has a 3′-5′-exonuclease activity, and in amore preferred embodiment, said additional catalytic domain is TREX,more preferably TREX2 catalytic domain (WO2012/058458). In anotherpreferred embodiment, said catalytic domain is encoded by a single chainTREX2 polypeptide. Said additional catalytic domain may be fused to anuclease fusion protein or chimeric protein according to the inventionoptionally by a peptide linker.

Endonucleolytic breaks are known to stimulate the rate of homologousrecombination. Thus, in another embodiment, the genetic modificationstep of the method further comprises a step of introduction into cellsof an exogeneous nucleic acid comprising at least a sequence homologousto a portion of the target nucleic acid sequence, such that homologousrecombination occurs between the target nucleic acid sequence and theexogeneous nucleic acid. In particular embodiments, said exogenousnucleic acid comprises first and second portions which are homologous toregion 5′ and 3′ of the target nucleic acid sequence, respectively. Saidexogenous nucleic acid in these embodiments also comprises a thirdportion positioned between the first and the second portion whichcomprises no homology with the regions 5′ and 3′ of the target nucleicacid sequence. Following cleavage of the target nucleic acid sequence, ahomologous recombination event is stimulated between the target nucleicacid sequence and the exogenous nucleic acid. Preferably, homologoussequences of at least 50 bp, preferably more than 100 bp and morepreferably more than 200 bp are used within said donor matrix. In aparticular embodiment, the homologous sequence can be from 200 bp to6000 bp, more preferably from 1000 bp to 2000 bp. Indeed, shared nucleicacid homologies are located in regions flanking upstream and downstreamthe site of the break and the nucleic acid sequence to be introducedshould be located between the two arms.

Immune Check Points

The present invention provides allogeneic T-cells expressing ananti-CD123 CAR, in particular an anti-CD123 CAR of SEQ ID N° 44, or ofSEQ ID N° 1+SEQ ID N° 44, wherein at least one gene expressing one ormore component of T-cell receptor (TCR) is inactivated and/or one geneselected from the genes CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1,LAG3, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9,CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD,FAS, TGFBRII, TGFBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1,IL10RA, IL10RB, HMOX2, IL6R, IL6ST, CSK, PAG1, SIT1, FOXP3, PRDM1(orblimp1), BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, is inactivated asreferred to in WO2014/184741.

Drug Resistant T-Cells

According to another aspect, the anti-CD123 CAR expressing T-cell of theinvention can be further genetically engineered to improve itsresistance to immunosuppressive drugs or chemotherapy treatments, whichare used as standard care for treating CD123 positive malignant cells.

Several cytotoxic agents (anti-cancer drugs) such as anti-metabolites,alkylating agents, anthracyclines, DNA methyltransferase inhibitors,platinum compounds and spindle poisons have been developed to killcancer cells. However, the introduction of these agents with noveltherapies, such as immunotherapies, is problematic. For example,chemotherapy agents can be detrimental to the establishment of robustanti-tumor immunocompetent cells due to the agents' non-specifictoxicity profiles. Small molecule-based therapies targeting cellproliferation pathways may also hamper the establishment of anti-tumorimmunity. If chemotherapy regimens that are transiently effective can becombined with novel immunocompetent cell therapies then significantimprovement in anti-neoplastic therapy might be achieved (for review(Dasgupta, McCarty et al. 2011)).

To improve cancer therapy and selective engraftment of allogeneicT-cells, drug resistance is conferred to said allogeneic T cells toprotect them from the toxic side effects of chemotherapy agent. The drugresistance of T-cells also permits their enrichment in or ex vivo, asT-cells which express the drug resistance gene will survive and multiplyrelative to drug sensitive cells.

Methods for engineering T-cells resistant to chemotherapeutic agents aredisclosed in PCT/EP2014/075317 which is fully incorporated by referenceherein.

In particular, the present invention relates to a method of engineeringallogeneic cells suitable for immunotherapy wherein at least one geneencoding a T-cell receptor (TCR) component is inactivated and one geneis modified to confer drug resistance comprising:

-   -   Providing an anti-CD123 CAR expressing T-cell; in particular an        anti-CD123 CAR of SEQ ID N0° 42, SEQ ID N0° 44, SEQ ID N0 46        expressing T cell, preferably humanized 123 CAR of SEQ ID N0°        42, SEQ ID N0° 44, SEQ ID NO 46    -   Modifying said anti-CD123 CAR expressing T-cell by inactivating        at least one gene encoding a T-cell receptor (TCR) component;    -   Modifying said anti-CD123 CAR expressing T-cell to confer drug        resistance to said anti-CD123 CAR expressing T-cell;    -   Expanding said engineered anti-CD123 CAR expressing T-cell in        the presence of said drug.

Alternatively, the present invention relates to a method comprising:

-   -   Providing an anti-CD123 CAR expressing T-cell; in particular an        anti-CD123 CAR of SEQ ID N° 42 SEQ ID N0° 44, SEQ ID N0 46        expressing T cell, preferably humanized 123 CAR of SEQ ID N0°        42, SEQ ID N0° 44, SEQ ID NO 46    -   Modifying said anti-CD123 CAR expressing T-cell to confer drug        resistance to said anti-CD123 CAR expressing T-cell;    -   Modifying said anti-CD123 CAR expressing T-cell by inactivating        at least one gene encoding a T-cell receptor (TCR) component;    -   Expanding said engineered anti-CD123 CAR expressing T-cell in        the presence of said drug.

In particular, the present invention also relates to a method ofengineering allogeneic cells suitable for immunotherapy wherein at leastone gene encoding a T-cell receptor (TCR) component is inactivated andone gene is modified to confer drug resistance comprising:

-   -   Providing an anti-CD123 CAR expressing T-cell; in particular an        anti-CD123 CAR of SEQ ID N0° 75, SEQ ID N0° 76, SEQ ID NO 77        expressing T cell, preferably humanized 123 CAR of SEQ ID N0°        75, SEQ ID N0° 76, SEQ ID NO 77, more preferably humanized 123        CAR of SEQ ID N0° 75,    -   Modifying said anti-CD123 CAR expressing T-cell by inactivating        at least one gene encoding a T-cell receptor (TCR) component;    -   Modifying said anti-CD123 CAR expressing T-cell to confer drug        resistance to said anti-CD123 CAR expressing T-cell;    -   Expanding said engineered anti-CD123 CAR expressing T-cell in        the presence of said drug.

Alternatively, the present invention relates to a method comprising:

-   -   Providing an anti-CD123 CAR expressing T-cell; in particular an        anti-CD123 CAR of SEQ ID N° 75, SEQ ID N0° 76, SEQ ID NO 77        expressing T cell, preferably humanized 123 CAR of SEQ ID N0°        75, SEQ ID N0° 76, SEQ ID NO 77, more preferably humanized 123        CAR of SEQ ID N0° 75,    -   Modifying said anti-CD123 CAR expressing T-cell to confer drug        resistance to said anti-CD123 CAR expressing T-cell;    -   Modifying said anti-CD123 CAR expressing T-cell by inactivating        at least one gene encoding a T-cell receptor (TCR) component;

Expanding said engineered anti-CD123 CAR expressing T-cell in thepresence of said drug.

Expression of Drug Resistance Genes in Anti-CD123 CAR-Expressing ImmuneCells

In a particular embodiment, said drug resistance can be conferred to theT-cell by the expression of at least one drug resistance gene. Said drugresistance gene refers to a nucleic acid sequence that encodes“resistance” to an agent, such as a chemotherapeutic agent (e.g.methotrexate). In other words, the expression of the drug resistancegene in a cell permits proliferation of the cells in the presence of theagent to a greater extent than the proliferation of a corresponding cellwithout the drug resistance gene. The expression of the drug resistancegene in a cell permits proliferation of the cells in the presence of theagent and does not affect its activity. A drug resistance gene of theinvention can encode resistance to anti-metabolite, methotrexate,vinblastine, cisplatin, alkylating agents, anthracyclines, cytotoxicantibiotics, anti-immunophilins, their analogs or derivatives, and thelike.

In one embodiment, a drug resistance gene of the invention can conferresistance to a drug (or an agent), in particular an anti-cancer drugselected from Aracytine, Cytosine Arabinoside, amsacrine, Daunorubicine,Idarubicine, Novantrone, Mitoxantrone, Vepeside, Etoposide (VP16),arsenic trioxyde, transretinoic acid, combination of arsenic trioxyde,transretinoic acid, mechlorethamine, procarbazine, chlorambucil,cytarabine, a nthracyclines, 6-thioguanine, hydroxyurea, prednisone, andcombination thereof.

Several drug resistance genes have been identified that can potentiallybe used to confer drug resistance to targeted cells (Takebe, Zhao et al.2001; Sugimoto, Tsukahara et al. 2003; Zielske, Reese et al. 2003;Nivens, Felder et al. 2004; Bardenheuer, Lehmberg et al. 2005; Kushman,Kabler et al. 2007).

One example of drug resistance gene can also be a mutant or modifiedform of Dihydrofolate reductase (DHFR). DHFR is an enzyme involved inregulating the amount of tetrahydrofolate in the cell and is essentialto DNA synthesis. Folate analogs such as methotrexate (MTX) inhibit DHFRand are thus used as anti-neoplastic agents in clinic. Different mutantforms of DHFR which have increased resistance to inhibition byanti-folates used in therapy have been described. In a particularembodiment, the drug resistance gene according to the present inventioncan be a nucleic acid sequence encoding a mutant form of human wild typeDHFR (GenBank: AAH71996.1) which comprises at least one mutationconferring resistance to an anti-folate treatment, such as methotrexate.In particular embodiment, mutant form of DHFR comprises at least onemutated amino acid at position G15, L22, F31 or F34, preferably atpositions L22 or F31 (Schweitzer, Dicker et al. 1990); Internationalapplication WO94/24277; U.S. Pat. No. 6,642,043). In a particularembodiment, said DHFR mutant form comprises two mutated amino acids atposition L22 and F31. Correspondence of amino acid positions describedherein is frequently expressed in terms of the positions of the aminoacids of the form of wild-type DHFR polypeptide set forth in GenBank:AAH71996.1. In a particular embodiment, the serine residue at position15 is preferably replaced with a tryptophan residue. In anotherparticular embodiment, the leucine residue at position 22 is preferablyreplaced with an amino acid which will disrupt binding of the mutantDHFR to antifolates, preferably with uncharged amino acid residues suchas phenylalanine or tyrosine. In another particular embodiment, thephenylalanine residue at positions 31 or 34 is preferably replaced witha small hydrophilic amino acid such as alanine, serine or glycine.

As used herein, “antifolate agent” or “folate analogs” refers to amolecule directed to interfere with the folate metabolic pathway at somelevel. Examples of antifolate agents include, e.g., methotrexate (MIX);aminopterin; trimetrexate (Neutrexin™); edatrexate;N10-propargyl-5,8-dideazafolic acid (CB3717); ZD1694 (Tumodex),5,8-dideazaisofolic acid (IAHQ); 5,10-dideazatetrahydrofolic acid(DDATHF); 5-deazafolic acid; PT523 (N alpha-(4-amino-4-deoxypteroyl)-Ndelta-hemiphthaloyl-L-ornithine); 10-ethyl-10-deazaaminopterin (DDATHF,lomatrexol); piritrexim; 10-EDAM; ZD1694; GW1843; Pemetrexate and PDX(10-propargyl-10-deazaaminopterin).

Another example of drug resistance gene can also be a mutant or modifiedform of ionisine-5′-monophosphate dehydrogenase II (IMPDH2), arate-limiting enzyme in the de novo synthesis of guanosine nucleotides.The mutant or modified form of IMPDH2 is an IMPDH inhibitor resistancegene. IMPDH inhibitors can be mycophenolic acid (MPA) or its prodrugmycophenolate mofetil (MMF). The mutant IMPDH2 can comprises at leastone, preferably two mutations in the MAP binding site of the wild typehuman IMPDH2 (NP_000875.2) that lead to a significantly increasedresistance to IMPDH inhibitor. The mutations are preferably at positionsT333 and/or S351 (Yam, Jensen et al. 2006; Sangiolo, Lesnikova et al.2007; Jonnalagadda, Brown et al. 2013). In a particular embodiment, thethreonine residue at position 333 is replaced with an isoleucine residueand the serine residue at position 351 is replaced with a tyrosineresidue. Correspondence of amino acid positions described herein isfrequently expressed in terms of the positions of the amino acids of theform of wild-type human IMPDH2 polypeptide set forth in NP_000875.2.

Another drug resistance gene is the mutant form of calcineurin.Calcineurin (PP2B), an ubiquitously expressed serine/threonine proteinphosphatase that is involved in many biological processes and which iscentral to T-cell activation. Calcineurin is a heterodimer composed of acatalytic subunit (CnA; three isoforms) and a regulatory subunit (CnB;two isoforms). After engagement of the T-cell receptor, calcineurindephosphorylates the transcription factor NFAT, allowing it totranslocate to the nucleus and active key target gene such as IL2. FK506in complex with FKBP12, or cyclosporine A (CsA) in complex with CyPAblock NFAT access to calcineurin's active site, preventing itsdephosphorylation and thereby inhibiting T-cell activation (Brewin,Mancao et al. 2009). The drug resistance gene of the present inventioncan be a nucleic acid sequence encoding a mutant form of calcineurinresistant to calcineurin inhibitor such as FK506 and/or CsA. In aparticular embodiment, said mutant form can comprise at least onemutated amino acid of the wild type calcineurin heterodimer a atpositions: V314, Y341, M347, T351, W352, L354, K360, preferably doublemutations at positions T351 and L354 or V314 and Y341. In a particularembodiment, the valine residue at position 341 can be replaced with alysine or an arginine residue, the tyrosine residue at position 341 canbe replaced with a phenylalanine residue; the methionine at position 347can be replaced with the glutamic acid, arginine or tryptophane residue;the threonine at position 351 can be replaced with the glutamic acidresidue; the tryptophane residue at position 352 can be replaced with acysteine, glutamic acid or alanine residue, the serine at position 353can be replaced with the histidine or asparagines residue, the leucineat position 354 can be replaced with an alanine residue; the lysine atposition 360 can be replaced with an alanine or phenylalanine residue ofa sequence corresponding to GenBank: ACX34092.1. Correspondence of aminoacid positions described herein is frequently expressed in terms of thepositions of the amino acids of the form of wild-type human calcineurinheterodimer a polypeptide set forth in (GenBank: ACX34092.1).

In another particular embodiment, said mutant form can comprise at leastone mutated amino acid of the wild type calcineurin heterodimer b atpositions: V120, N123, L124 or K125, preferably double mutations atpositions L124 and K125. In a particular embodiment, the valine atposition 120 can be replaced with a serine, an aspartic acid,phenylalanine or leucine residue; the asparagine at position 123 can bereplaced with a tryptophan, lysine, phenylalanine, arginine, histidineor serine; the leucine at position 124 can be replaced with a threonineresidue; the lysine at position 125 can be replaced with an alanine, aglutamic acid, tryptophan, or two residues such as leucine-arginine orisoleucine-glutamic acid can be added after the lysine at position 125in the amino acid sequence corresponding to GenBank: ACX34095.1.Correspondence of amino acid positions described herein is frequentlyexpressed in terms of the positions of the amino acids of the form ofwild-type human calcineurin heterodimer b polypeptide set forth in(GenBank: ACX34095.1).

Another drug resistance gene is 0(6)-methylguanine methyltransferase(MGMT) encoding human alkyl guanine transferase (hAGT). AGT is a DNArepair protein that confers resistance to the cytotoxic effects ofalkylating agents, such as nitrosoureas and temozolomide (TMZ).6-benzylguanine (6-BG) is an inhibitor of AGT that potentiatesnitrosourea toxicity and is co-administered with TMZ to potentiate thecytotoxic effects of this agent. Several mutant forms of MGMT thatencode variants of AGT are highly resistant to inactivation by 6-BG, butretain their ability to repair DNA damage (Maze, Kurpad et al. 1999). Ina particular embodiment, AGT mutant form can comprise a mutated aminoacid of the wild type AGT position P140, in the amino acid sequence SEQID NO: 18 (UniProtKB: P16455). In a preferred embodiment, said prolineat position 140 is replaced with a lysine residue.

Another drug resistance gene can be multidrug resistance protein 1(MDR1) gene. This gene encodes a membrane glycoprotein, known asP-glycoprotein (P-GP) involved in the transport of metabolic byproductsacross the cell membrane. The P-Gp protein displays broad specificitytowards several structurally unrelated chemotherapy agents.

Overexpressing multidrug resistance protein 1 has been described toconfer resistance to drugs such as Mitoxantrone (Charles S. Morrow,Christina Peklak-Scott, Bimjhana Bishwokarma, Timothy E. Kute, Pamela K.Smitherman, and Alan J. Townsend. Multidrug Resistance Protein 1 (MRP1,ABCC1) Mediates Resistance to Mitoxantrone via Glutathione-DependentDrug Efflux Mol Pharmacol April 2006 69:1499-1505).

Thus, drug resistance can be conferred to cells by the expression ofnucleic acid sequence that encodes MDR-1 (NP_000918).

Still another way of preparing drug resistant cells is to prepare cellswith specific mutation (s) such as mutations at Arg486 and Glu571 in theHuman Topoisomerase II gene, to confer resistance to amsacrine (S.PATEL, B. A. KELLER, and L. M. FISHER. 2000. MOLECULAR PHARMACOLOGY. Vol57: p 784-791 (2000).

Still another way of preparing drug resistant cells is to prepare cellsoverexpressing microRNA-21 to confer resistance to Daunorubicine(Involvement of miR-21 in resistance to daunorubicin by regulating PTENexpression in the leukaemia K562 cell line Bai, Haitao et al. FEBSLetters, Volume 585, Issue 2, 402-408).

In a preferred embodiment, cells bearing such a drug resistanceconferring mRNA or protein also comprise an inhibitory mRNA or a genethe expression of which is conditioned, allowing the selectivedestruction of said drug resistant cells in the presence of said drug orupon administration of said drug.

Drug resistance gene can also confer resistance to cytotoxicantibiotics, and can be ble gene or mcrA gene. Ectopic expression of blegene or mcrA in an immune cell gives a selective advantage when exposedto the chemotherapeutic agent, respectively the bleomycine or themitomycin C.

The most practical approach to gene therapy is the addition of a gene toengineer T-cell by using efficient gene delivery with vectors,preferably viral vector. Thus, in a particular embodiment, said drugresistance gene can be expressed in the cell by introducing a transgenepreferably encoded by at least one vector into a cell.

In one embodiment, cells bearing a drug resistance gene or a modifiedgene conferring resistance to a drug also comprise an inducible suicidegene—the induction of which provokes cell death—allowing their selectivedestruction.

The random insertion of genes into the genome may lead to theinappropriate expression of the inserted gene or the gene near theinsertion site. Specific gene therapy using homologous recombination ofexogenous nucleic acid comprising endogenous sequences to target genesto specific sites within the genome can allow engineering secureT-cells. As described above, the genetic modification step of the methodcan comprise a step of introduction into cells of an exogeneous nucleicacid comprising at least a sequence encoding the drug resistance geneand a portion of an endogenous gene such that homologous recombinationoccurs between the endogenous gene and the exogeneous nucleic acid. In aparticular embodiment, said endogenous gene can be the wild type “drugresistance” gene, such that after homologous recombination, the wildtype gene is replaced by the mutant form of the gene which confersresistance to the drug.

Endonucleolytic breaks are known to stimulate the rate of homologousrecombination. Thus, in a particular embodiment, the method of theinvention further comprises the step of expressing in the cell arare-cutting endonuclease which is able to cleave a target sequencewithin an endogenous gene. Said endogenous gene can encode for examplesDHFR, IMPDH2, calcineurin or AGT. Said rare-cutting endonuclease can bea TALE-nuclease, a Zinc finger nuclease, a CRISPR/Cas9 endonuclease, aMBBBD-nuclease or a meganuclease.

Inactivation of Drug Sensitizing Genes in Anti-CD123 CAR-ExpressingImmune Cells

In another particular embodiment, said drug resistance can be conferredto the cell of the invention (anti-CD123 CAR expressing immune cell,) bythe inactivation of a drug sensitizing gene.

The inventor sought to inactivate potential drug sensitizing gene toengineer T-cell for immunotherapy, in particular to engineer anti-CD123CAR expressing immune cell that can be used in combination with atherapeutic agent (anti-cancer drug).

By inactivating a gene it is intended that the gene of interest is notexpressed in a functional protein form. In particular embodiment, thegenetic modification of the method relies on the expression, in providedcells to engineer, of one rare-cutting endonuclease such that saidrare-cutting endonuclease specifically catalyzes cleavage in onetargeted gene thereby inactivating said targeted gene. In a particularembodiment, the step of inactivating at least one drug sensitizing genecomprises introducing into the cell a rare-cutting endonuclease able todisrupt at least one drug sensitizing gene. In a more particularembodiment, said cells are transformed with nucleic acid encoding arare-cutting endonuclease capable of disrupting a drug sensitizing gene,and said rare-cutting endonuclease is expressed into said cells. Saidrare-cutting endonuclease can be a meganuclease, a Zinc finger nuclease,CRISPR/Cas9 nuclease, A MBBBD-nuclease or a TALE-nuclease. In apreferred embodiment, said rare-cutting endonuclease is a TALE-nuclease.

In a preferred embodiment, drug sensitizing gene which can beinactivated to confer drug resistance to the T-cell is the humandeoxycytidine kinase (dCK) gene. This enzyme is required for thephosphorylation of the deoxyribonucleosides deoxycytidine (dC),deoxyguanosine (dG) and deoxyadenosine (dA). Purine nucleotide analogs(PNAs) are metabolized by dCK into mono-, di- and tri-phosphate PNA.Their triphosphate forms and particularly clofarabine triphosphatecompete with ATP for DNA synthesis, acts as proapoptotic agent and arepotent inhibitors of ribonucleotide reductase (RNR) which is involved intrinucleotide production.

Preferably, the inactivation of dCK in T cells is mediated by TALEnuclease. To achieve this goal, several pairs of dCK TALE-nuclease havebeen designed, assembled at the polynucleotide level and validated bysequencing. Examples of TALE-nuclease pairs which can be used accordingto the invention are depicted in PCT/EP2014/075317.

This dCK inactivation in T cells confers resistance to purine nucleosideanalogs (PNAs) such as clofarabine and fludarabine.

In another preferred embodiment, the dCK inactivation in T cells iscombined with an inactivation of TRAC genes rendering these double knockout (KO) T cells both resistant to drug such as clofarabine and lessallogeneic. This double features is particularly useful for atherapeutic goal, allowing “off-the-shelf” allogeneic cells forimmunotherapy in conjunction with chemotherapy to treat patients withcancer. This double KO inactivation dCK/TRAC can be performedsimultaneously or sequentially. One example of TALE-nuclease dCK/TRACpairs which gave success in the invention is described inPCT/EP2014/075317, in particular, the target sequences in the 2 loci(dCK and TRAC).

Another example of enzyme which can be inactivated is humanhypoxanthine-guanine phosphoribosyl transferase (HPRT) gene (Genbank:M26434.1). In particular HPRT can be inactivated in engineered T-cellsto confer resistance to a cytostatic metabolite, the 6-thioguanine (6TG)which is converted by HPRT to cytotoxic thioguanine nucleotide and whichis currently used to treat patients with cancer, in particular leukemias(Hacke, Treger et al. 2013). Guanines analogs are metabolized by HPRTtransferase that catalyzes addition of phosphoribosyl moiety and enablesthe formation of TGMP Guanine analogues including 6 mercapthopurine(6MP) and 6 thioguanine (6TG) are usually used as lymphodepleting drugsto treat ALL. They are metabolized by HPRT (hypoxanthine phosphoribosyltransferase that catalyzes addition of phosphoribosyl moiety and enablesformation TGMP. Their subsequent phosphorylations lead to the formationof their triphosphorylated forms that are eventually integrated intoDNA. Once incorporated into DNA, thio GTP impairs fidelity of DNAreplication via its thiolate groupment and generate random pointmutation that are highly deleterious for cell integrity.

In another embodiment, the inactivation of the CD3 normally expressed atthe surface of the T-cell can confer resistance to anti-CD3 antibodiessuch as teplizumab.

The terms “therapeutic agent”, “chemotherapeutic agent”, or “drug” or“anti-cancer drug” as used herein refers to a medicament, preferably acompound or a derivative thereof that can interact with a cancer cell,thereby reducing the proliferative status of the cell and/or killing thecell. Examples of chemotherapeutic agents or “anti-cancer drug” include,but are not limited to, alkylating agents (e.g.,Busulfan•Carboplatine•Chlorambucil•Cisplatine•Cyclophosphamide•Ifosfamide•Melphalan•Méchloréthamine•Oxaliplatine•Uramustine•Temozolomide•Fotemustine), metabolic antagonists (e.g.,purine nucleoside antimetabolite such as clofarabine, fludarabine or2′-deoxyadenosine, methotrexate (MTX), 5-fluorouracil or derivativesthereof,Azathioprine•Capecitabine•Cytarabine•Floxuridine•Fluorouracile•Gemcitabine•Methotrexate•Pemetrexed),antitumor antibiotics (e.g., mitomycin, Adriamycin,Bleomycine•Daunorubicine•Doxorubicine•Epirubicine•Hydroxyurea•Idarubicine•MitomycinC•Mitoxantrone), plant-derived antitumor agents (e.g., vincristine,vindesine, Taxol, Vinblastine•(Vinorelbine) •Docetaxel•Paclitaxel),topoisomerase inhibitor (Irinotecan•Topotecan•Etoposide),

In a preferred embodiment, a therapeutic agent, a chemotherapy drug asused herein refers to a compound or a derivative thereof that may beused to treat cancer, in particular to treat a hematopoietic cancer celland more particularly AML, thereby reducing the proliferative status ofthe cancer cell and/or killing the cancer cell. Examples ofchemotherapeutic agents include, but are not limited to Aracytine,Cytosine Arabinoside, Amsacrine, Daunorubicine, Idarubicine, Novantrone,Mitoxantrone, Vepeside, Etoposide (VP16), arsenic trioxyde,transretinoic acid, mechlorethamine, procarbazine, chlorambucil, andcombination thereof.

In other embodiments of the present invention, cells of the inventionare administered to a patient in conjunction with a drug (or an agent)selected from Aracytine, Cytosine Arabinoside, amsacrine, Daunorubicine,Idarubicine, Novantrone, Mitoxantrone, Vepeside, Etoposide (VP16),arsenic trioxyde, transretinoic acid, cytarabine, anthracyclines,6-thioguanine, hydroxyurea, prednisone, and combination thereof.

Such agents may further include, but are not limited to, the anti-canceragents TRIMETHOTRIXATE™ (TMTX), TEMOZOLOMIDE™, RALTRITREXED™,S-(4-Nitrobenzyl)-6-thioinosine (NBMPR),6-benzyguanidine (6-BG),bis-chloronitrosourea (BCNU) and CAMPTOTHECIN™, or a therapeuticderivative of any thereof.

In a more preferred embodiment an anti-CD123 CAR of SEQ ID N° 44expressing T cell, is administered to a patient, in combination with atleast one therapeutic agent selected from Aracytine, CytosineArabinoside, Amsacrine, Daunorubicine, Idarubicine, Novantrone,Mitoxantrone, Vepeside, Etoposide (VP16), arsenic trioxyde,transretinoic acid and combination thereof.

As used herein, a cell which is “resistant or tolerant” to an agentmeans a cell which has been genetically modified so that the cellproliferates in the presence of an amount of an agent that inhibits orprevents proliferation of a cell without the modification.

Multiple Drug Resistance of Anti-CD123 CAR-Expressing Immune Cells

In another particular embodiment, the inventors sought to develop an“off-the shelf” immunotherapy strategy, using allogeneic T-cells, inparticular allogenic anti-CD123 CAR expressing T-cell resistant tomultiple drugs to mediate selection of engineered T-cells when thepatient is treated with different drugs. The therapeutic efficiency canbe significantly enhanced by genetically engineering multiple drugresistance allogeneic T-cells. Such a strategy can be particularlyeffective in treating tumors that respond to drug combinations thatexhibit synergistic effects. Moreover multiple resistant engineeredT-cells can expand and be selected using minimal dose of drug agents.

Thus, the method according to the present invention can comprisemodifying T-cell to confer multiple drug resistance to said T-cell. Saidmultiple drug resistance can be conferred by either expressing more thanone drug resistance gene or by inactivating more than one drugsensitizing gene. In another particular embodiment, the multiple drugresistance can be conferred to said T-cell by expressing at least onedrug resistance gene and inactivating at least one drug sensitizinggene. In particular, the multiple drug resistance can be conferred tosaid T-cell by expressing at least one drug resistance gene such asmutant form of DHFR, mutant form of IMPDH2, mutant form of calcineurin,mutant form of MGMT, the ble gene, and the mcrA gene and inactivating atleast one drug sensitizing gene such as HPRT gene. In a preferredembodiment, multiple drug resistance can be conferred by inactivatingHPRT gene and expressing a mutant form of DHFR; or by inactivating HPRTgene and expressing a mutant form of IMPDH2; or by inactivating HPRTgene and expressing a mutant form of calcineurin; by inactivating HPRTgene and expressing a mutant form of MGMT; by inactivating HPRT gene andexpressing the ble gene; by inactivating HPRT gene and expressing themcrA gene.

In one embodiment, the present invention provides allogenic anti-CD123CAR expressing T-cell expressing more than one drug resistance gene orwherein more than one drug sensitizing gene is inactivated.

Suicide Genes in Anti-CD123 CAR-Expressing Immune Cells

In some instances, since engineered T-cells can expand and persist foryears after administration, it can be desirable to include a safetymechanism to allow selective deletion of administrated T-cells. Thus, insome embodiments, the method of the invention can comprises thetransformation of said T-cells with a recombinant suicide gene. Saidrecombinant suicide gene is used to reduce the risk of direct toxicityand/or uncontrolled proliferation of said T-cells once administrated ina subject (Quintarelli C, Vera F, blood 2007; Tey S K, Dotti G., RooneyC M, boil blood marrow transplant 2007). Suicide genes enable selectivedeletion of transformed cells in vivo. In particular, the suicide genehas the ability to convert a non-toxic pro-drug into cytotoxic drug orto express the toxic gene expression product. In other words, “Suicidegene” is a nucleic acid coding for a product, wherein the product causescell death by itself or in the presence of other compounds.

A representative example of such a suicide gene is one which codes forthymidine kinase of herpes simplex virus. Additional examples arethymidine kinase of varicella zoster virus and the bacterial genecytosine deaminase which can convert 5-fluorocytosine to the highlytoxic compound 5-fluorouracil. Suicide genes also include as nonlimiting examples caspase-9 or caspase-8 or cytosine deaminase.Caspase-9 can be activated using a specific chemical inducer ofdimerization (CID). Suicide genes can also be polypeptides that areexpressed at the surface of the cell and can make the cells sensitive totherapeutic monoclonal antibodies. As used herein “prodrug” means anycompound useful in the methods of the present invention that can beconverted to a toxic product. The prodrug is converted to a toxicproduct by the gene product of the suicide gene in the method of thepresent invention. A representative example of such a prodrug isganciclovir which is converted in vivo to a toxic compound byHSV-thymidine kinase. The ganciclovir derivative subsequently is toxicto tumor cells. Other representative examples of prodrugs includeacyclovir, FIAU[1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-5-iodouracil],6-methoxypurine arabinoside for VZV-TK, and 5-fluorocytosine forcytosine deaminase.

One preferred suicide gene system employs a recombinant antigenicpolypeptide comprising antigenic motif recognized by the anti-CD20 mAbRituximab, especially QBen10, such as in the so-called RQR8 polypeptidedescribed in WO2013153391. Rituximab, an authorized antibody drug, canthen be used for cell depletion when needed.

In one embodiment, the present invention provides allogenic anti-CD123CAR expressing T-cell expressing more than one drug resistance gene orwherein more than one drug sensitizing gene is inactivated, and asuicide gene allowing said cells to be destroyed.

Clofarabine Resistant Anti-CD123 CAR-Expressing Immune Cells

The invention encompasses the manufacture of T cells for therapeuticuse, which are resistant a drug such as to Clofarabine. They can beobtained by inactivation of the dCK gene such as previously explained.According to a preferred embodiment, the T-cells are made resistant tochemotherapy and less allogeneic by combining inactivation of dCK andTCR genes as previously described.

Thus, the present invention provides an anti-CD123 CAR expressing cell,in particular an anti-CD123 CAR expressing T cell wherein the CAR isderived from Klon 43 (comprising a SEQ ID N042 or SEQ ID NO. 44,optionally humanized) and wherein the dCK gene is inactivated.

CD123+/Luc+ Drug Resistant Daudi Cells for Testing the Cytotoxicity ofDrug Resistant Allogenic CART Cells

The present invention encompasses also a method for manufacturing targetcells which express both a surface receptor specific to the CART cellsand a resistance gene. These target cells are particularly useful fortesting the cytotoxicity of CAR T cells. These cells are readilyresistant to clinically relevant dose of clofarabine and harborluciferase activity. This combination of features enable traking them invivo in a mice model or destroy them when required.

More particularly, they can be used to assess the cytotoxicityproperties drug resistant T cells in mice in the presence of clofarabineor other PNAs. Clofarabine resistant Daudi cells mimick thephysiological state of acute lymphoblastic leukemia (ALL) patientsrelapsing form induction therapy, that harbor drug resistant B cellmalignancies. Thus, these cells are of great interest to evaluate thereliability and cytotoxicity of drug resistant CART cells. Preferably,these target cells are CD123+ Luciferase+ Daudi cells.

Isolated Cell

The present invention relates to an isolated cell expressing a CAR whichbinds to CD123. Thus, the invention relates to an anti-CD123 CARexpressing cell. In a particular embodiment, said anti-CD123 CARexpressing cell is resistant to at least one drug and/or comprises atleast one disrupted gene encoding a T-cell receptor component.

In a preferred embodiment, the present invention relates to an isolatedT cell expressing a CAR which binds to CD123. The invention relates toan anti-CD123 CAR expressing T-cell. In a particular embodiment, saidanti-CD123 CAR expressing T cell is resistant to at least one drugand/or comprises at least one disrupted gene encoding a T-cell receptorcomponent.

In a particular embodiment, said anti-CD123 CAR T-cell expresses atleast one drug resistance gene, preferably ble gene or mcrA gene or geneencoding a mutant DHFR, a mutant IMPDH2, a mutant AGT or a mutantcalcineurin.

In another particular embodiment, said anti-CD123 CAR expressing T cellcomprises at least one disrupted drug sensitizing gene such as dCK orHPRT gene. In a more particular embodiment, said isolated anti-CD123 CART-cell comprises a disrupted HPRT gene and express a DHFR mutant; saidisolated anti-CD123 CAR T-cell comprises a disrupted HPRT gene andexpress a IMPDH2 mutant; said isolated anti-CD123 CAR T-cell comprises adisrupted HPRT gene and express a calcineurin mutant; said isolatedanti-CD123 CAR T-cell comprises a disrupted HPRT gene and express a AGTmutant.

Allogeneic Anti-CD123 CAR T-Cell Resistant to a Drug for its Use inImmunotherapy

In particular, the present invention relates to an allogeneic T-cell, inparticular an allogeneic anti-CD123 CAR expressing T-cell, andpreferably an allogeneic anti-CD123 CAR expressing T-cell comprising apeptide having 80% to 100% identity with scfv from Klon 43, saidallogeneic anti-CD123 CAR expressing T-cell comprising a peptide having80% to 100% identity with scfv from Klon 43 is more particularlyresistant to a drug, and specifically suitable for immunotherapy.

In a preferred embodiment, said allogeneic anti-CD123 CAR expressingT-cell comprises a peptide having 80% to 100% identity with SEQ ID NO.44and is more particularly resistant to a drug, and specifically suitablefor immunotherapy.

The resistance of a drug can be conferred by inactivation of drugsensitizing genes or by expression of drug resistance genes. Someexamples of drugs which suit to the invention are the purine nucleosideanalogues (PNAs) such as clofarabine or fludarabine, or other drugs suchas 6-Mercaptopurine (6MP) and 6 thio-guanine (6TG).

In one aspect, the present invention provides methods for engineeringimmune cells to make them resistant to purine nucleotide analogs (PNA),such a clorofarabine or fludarabine, so that they can be used in cancerimmunotherapy treatments in patients pre-treated with these conventionalchemotherapies.

The resistance to drugs can be conferred to the T-cells by inactivatingone or more gene(s) responsible for the cell's sensitivity to the drug(drug sensitizing gene(s)), such as the dcK and/or HPRT genes.

According to another aspect, the resistance to drugs can be conferred toa T-cell by expressing a drug resistance gene. Variant alleles ofseveral genes such as dihydrofolate reductase (DHFR), inosinemonophosphate dehydrogenase 2 (IMPDH2), calcineurin or methylguaninetransferase (MGMT) have been identified to confer drug resistance to acell according to the invention.

For instance, CD52 and glucocorticoid receptors (GR), which are drugtargets of Campath (alemtuzumab) or rituximab and glucocorticoidstreatments, can be inactivated to make the cells resistant to thesetreatments and give them a competitive advantage over patient's ownT-cells not endowed with specific CD123 CARs. Expression of CD3 gene canalso be suppressed or reduced to confer resistance to Teplizumab, whichis another immune suppressive drug. Expression of HPRT can also besuppressed or reduced according to the invention to confer resistance to6-thioguanine, a cytostatic agent commonly used in chemotherapyespecially for the treatment of acute lymphoblasic leukemia.

According to further aspect of the invention, the immune cells can befurther manipulated to make them more active or limit exhaustion, byinactivating genes encoding proteins that act as “immune checkpoints”that act as regulators of T-cells activation, such as the following geneselected from CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, LAG3, HAVCR2,BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244,TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS,TGFBRII, TGFBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA,IL10RB, HMOX2, IL6R, IL6ST, CSK, PAG1, SIT1, FOXP3, PRDM1 (orblimp1),BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, preferably, said gene is PDCD1or CTLA-4. Examples of genes, which expression could be reduced orsuppressed are indicated in Table 9.

In one embodiment said gene is a gene that acts as a regulator ofT-cells activation coding the beta 2 microglobulin protein.

According to a further aspect of the invention, the anti-CD123CAR-immune cells of the invention can be further manipulated to makethem resistant to a drug, in particular to a drug used duringchemotherapy against cancer, in particular a CD123-expressingcell-mediated cancer such as AML. This can be achieved by introducing agene conferring resistance to said drug. This same gene may be turned onand off by using a gene inducible inhibition/expression system aspreviously described (Garcia E L, Mills A A (2002) Getting aroundlethality with inducible Cre-mediated excision. Semin Cell Dev Biol13:151-8, Lewandoski M (2001) Conditional control of gene expression inthe mouse. Nat Rev Genet 2:743-55; Scharfenberger L, Hennerici T, KirlyG et al. (2014) Transgenic mouse technology in skin biology: Generationof complete or tissue-specific knockout mice. J Invest Dermatol 134:e16;Schwenk F, Kuhn R, Angrand P O et al. (1998) Temporally and spatiallyregulated somatic mutagenesis in mice. Nucleic Acids Res 26:1427-32

Thus, anti-CD123 CAR-expressing, drug resistant immune cell, wherein (i)at least one gene expressing one or more component of T-cell receptor(TCR) is inactivated (ii) at least one gene conferring resistance to adrug is incorporated or a gene conferring sensitivity to said drug isdeleted or mutated to be inactivated (iii) optionally another geneselected from the gene disclosed in table 9 is inactivated—is an objectof the present invention.

The present invention encompasses the isolated anti-CD123 CAR-immunecells or cell lines obtainable by the method of the invention, moreparticularly isolated cells comprising any of the proteins,polypeptides, allelic variants, altered or deleted genes or vectorsdescribed herein.

The immune cells of the present invention or cell lines can furthercomprise exogenous recombinant polynucleotides, in particular CARs orsuicide genes or they can comprise altered or deleted genes coding forcheckpoint proteins or ligands thereof that contribute to theirefficiency as a therapeutic product, ideally as an “off the shelf”product. In another aspect, the present invention concerns the methodfor treating or preventing cancer in the patient by administrating atleast once an engineered immune cell obtainable by the above methods.

TABLE 9 List of genes encoding immune checkpoint proteins. Genes thatcan be inactivated Pathway In the pathway Co-inhibitory CTLA4 (CD152)CTLA4, PPP2CA, PPP2CB, receptors PTPN6, PTPN22 PDCD1 (PD-1, CD279) PDCD1CD223 (lag3) LAG3 HAVCR2 (tim3) HAVCR2 BTLA(cd272) BTLA CD160(by55)CD160 IgSF family TIGIT CD96 CRTAM LAIR1(cd305) LAIR1 SIGLECs SIGLEC7SIGLEC9 CD244(2b4) CD244 Death receptors TRAIL TNFRSF10B, TNFRSF10A,CASP8, CASP10, CASP3, CASP6, CASP7 FAS FADD, FAS Cytokine TGF-betasignaling TGFBRII, TGFBRI, SMAD2, signalling SMAD3, SMAD4, SMAD10, SKI,SKIL, TGIF1 IL10 signalling IL10RA, IL10RB, HMOX2 IL6 signalling IL6R,IL6ST Prevention of CSK, PAG1 TCR signalling SIT1 Induced Treg inducedTreg FOXP3 Transcription transcription factors PRDM1 (=blimp1, factorscontrolling exhaustion heterozygotes mice control controlling chronicviral infection better exhaustion than wt or conditional KO) BATFHypoxia iNOS induced GUCY1A2, GUCY1A3, mediated guanylated cyclaseGUCY1B2, GUCY1B3 tolerance

TABLE 10 Sequence of the different humanized antibody fragmentsFunctional domains SEQ ID # Raw amino acid sequence Humanized scFv SEQ ID  MADYKDIVMTQSPSSVSASVGDRVTITCRA Klon43 Variant VL1 NO. 54SQNVDSAVAWYQQKPGKAPKALIYSASYRY SGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKR Humanized scFv  SEQ ID MADYKDIQMTQSPSSVSASVGDRVTITCRA Klon43 Variant VL2 NO. 55SQNVDSAVAWYQQKPGKAPKALIYSASYRY SGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKR Humanized scFv  SEQ ID MADYKDIQMTQSPSSVSASVGDRVTITCRA Klon43 Variant VL3 NO. 56SQNVDSAVAWYQQKPGKAPKALIYSASYRY SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKR Humanized scFv  SEQ ID MADYKDIQMTQSPSSVSASVGDRVTITCRA Klon43 Variant VL4 NO. 57SQNVDSAVAWYQQKPGKAPKLLIYSASYRY SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKR Humanized scFv  SEQ ID MADYKDIQMTQSPSSVSASVGDRVTITCRA Klon43V ariant VL5 NO. 58SQNVDSAVAWYQQKPGKAPKLLIYSASYRQ SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKR Humanized scFv  SEQ ID MADYKDIQMTQSPSSVSASVGDRVTITCRA Klon43 Variant VL6 NO. 59SQNVDSAVAWYQQKPGKAPKLLIYSASYGQ SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKR Humanized scFv  SEQ ID EVKLVESGGGLVQPGRSLRLSCTASGFTFTDY Klon43 Variant VH1 NO. 60YMSWVRQAPGKGLEWVGLIRSKADGYTTEYSAS VKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS Humanized scFv  SEQ ID EVQLVESGGGLVQPGRSLRLSCTASGFTFTDY Klon43 Variant VH2 NO. 61YMSWVRQAPGKGLEWVGLIRSKADGYTTEYSA SVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS Humanized scFv  SEQ ID EVQLVESGGGLVQPGRSLRLSCTASGFTFTDY Klon43 Variant VH3 NO. 62YMSWVRQAPGKGLEWVGLIRSKADGYTTEYSAS VKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS Humanized scFv  SEQ ID EVQLVESGGGLVQPGRSLRLSCTASGFTFTDY Klon43 Variant VH4 NO. 63YMSWVRQAPGKGLEWVGFIRSKADGYTTEYSAS VKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS Humanized scFv  SEQ ID EVQLVESGGGLVQPGRSLRLSCTASGFTFTDY Klon43 Variant VH5 NO. 64YMSWVRQAPGKGLEWVGFIRSKADGYTTEYAAS VKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS Humanized scFv  SEQ ID EVQLVESGGGLVQPGRSLRLSCTASGFTFTDY Klon43 Variant VH6 NO. 65YMSWVRQAPGKGLEWVGLIRSKADGYTTEYAA SVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS Humanized scFv  SEQ ID EVQLVESGGGLVQPGRSLRLSCTASGFTFTD Klon43 Variant VH7 NO. 66YYMSWVRQAPGKGLEWVGFIRSKADGYTT EYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRDAAYYSYYSPEGAMDYWGQG TLVTVSS

In a preferred embodiment said method of further engineer the immunecells involves introducing into said T cells polynucleotides, inparticular mRNAs, encoding specific rare-cutting endonuclease toselectively inactivate the genes mentioned above by DNA cleavage. In amore preferred embodiment said rare-cutting endonucleases areTALE-nucleases or Cas9 endonuclease. TAL-nucleases have so far provenhigher specificity and cleavage efficiency over the other types ofrare-cutting endonucleases, making them the endonucleases of choice forproducing of the engineered immune cells on a large scale with aconstant turn-over.

Delivery Methods

The different methods described above involve expressing a protein ofinterest such as drug resistance gene, rare-cutting endonuclease,Chimeric Antigen Receptor (CAR), in particular an anti-CD123 CAR andmore particularly, a CAR comprising a SEQ ID NO. 1+SEQ ID NO. 44, and asuicide gene into a cell.

As non-limiting example, said protein of interest can be expressed inthe cell by its introduction as a transgene preferably encoded by atleast one plasmid vector. Polypeptides may be expressed in the cell as aresult of the introduction of polynucleotides encoding said polypeptidesinto the cell. Alternatively, said polypeptides could be producedoutside the cell and then introduced thereto.

Methods for introducing a polynucleotide construct into cells are knownin the art and include as non limiting examples stable transformationmethods wherein the polynucleotide construct is integrated into thegenome of the cell, transient transformation methods wherein thepolynucleotide construct is not integrated into the genome of the celland virus mediated methods.

Said polynucleotides may be introduced into a cell by for example,recombinant viral vectors (e.g. retroviruses, adenoviruses), liposomeand the like. For example, transient transformation methods include forexample microinjection, electroporation or particle bombardment, cellfusion. Said polynucleotides may be included in vectors, moreparticularly plasmids or virus, in view of being expressed in cells.Said plasmid vector can comprise a selection marker which provides foridentification and/or selection of cells which received said vector.

Different transgenes can be included in one vector. Said vector cancomprise a nucleic acid sequence encoding ribosomal skip sequence suchas a sequence encoding a 2A peptide. 2A peptides, which were identifiedin the Aphthovirus subgroup of picornaviruses, causes a ribosomal “skip”from one codon to the next without the formation of a peptide bondbetween the two amino acids encoded by the codons (see Donnelly et al.,J. of General Virology 82: 1013-1025 (2001); Donnelly et al., J. of Gen.Virology 78: 13-21 (1997); Doronina et al., Mol. And. Cell. Biology28(13): 4227-4239 (2008); Atkins et al., RNA 13: 803-810 (2007)).

By “codon” is meant three nucleotides on an mRNA (or on the sense strandof a DNA molecule) that are translated by a ribosome into one amino acidresidue. Thus, two polypeptides can be synthesized from a single,contiguous open reading frame within an mRNA when the polypeptides areseparated by a 2A oligopeptide sequence that is in frame. Such ribosomalskip mechanisms are well known in the art and are known to be used byseveral vectors for the expression of several proteins encoded by asingle messenger RNA.

In a more preferred embodiment of the invention, polynucleotidesencoding polypeptides according to the present invention can be mRNAwhich is introduced directly into the cells, for example byelectroporation. The inventors determined the optimal condition for mRNAelectroporation in T-cell. The inventor used the cytoPulse technologywhich allows, by the use of pulsed electric fields, to transientlypermeabilize living cells for delivery of material into the cells. Thetechnology, based on the use of PulseAgile (BTX Havard Apparatus, 84October Hill Road, Holliston, Mass. 01746, USA) electroporationwaveforms grants the precise control of pulse duration, intensity aswell as the interval between pulses (U.S. Pat. No. 6,010,613 andInternational PCT application WO2004083379). All these parameters can bemodified in order to reach the best conditions for high transfectionefficiency with minimal mortality. Basically, the first high electricfield pulses allow pore formation, while subsequent lower electric fieldpulses allow moving the polynucleotide into the cell.

The different methods described above involve introducing CAR into acell. As non-limiting example, said CAR can be introduced as transgenesencoded by one plasmid vector. Said plasmid vector can also contain aselection marker which provides for identification and/or selection ofcells which received said vector.

Polypeptides may be synthesized in situ in the cell as a result of theintroduction of polynucleotides encoding said polypeptides into thecell. Alternatively, said polypeptides could be produced outside thecell and then introduced thereto. Methods for introducing apolynucleotide construct into cells are known in the art and includingas non limiting examples stable transformation methods wherein thepolynucleotide construct is integrated into the genome of the cell,transient transformation methods wherein the polynucleotide construct isnot integrated into the genome of the cell and virus mediated methods.Said polynucleotides may be introduced into a cell by for example,recombinant viral vectors (e.g. retroviruses, adenoviruses), liposomeand the like. For example, transient transformation methods include forexample microinjection, electroporation or particle bombardment. Saidpolynucleotides may be included in vectors, more particularly plasmidsor virus, in view of being expressed in cells.

Engineered Immune Cells

The present invention also relates to isolated cells or cell linessusceptible to be obtained by said method to engineer cells. Inparticular said isolated cell comprises at least one CAR as describedabove. In another embodiment, said isolated cell comprises a populationof CARs each one comprising different extracellular ligand bindingdomains. In particular, said isolated cell comprises exogenouspolynucleotide sequence encoding CAR. Genetically modified immune cellsof the present invention are activated and proliferate independently ofantigen binding mechanisms.

In the scope of the present invention is also encompassed an isolatedimmune cell, preferably a T-cell obtained according to any one of themethods previously described. Said immune cell refers to a cell ofhematopoietic origin functionally involved in the initiation and/orexecution of innate and/or adaptative immune response. Said immune cellaccording to the present invention can be derived from a stem cell. Thestem cells can be adult stem cells, non-human embryonic stem cells, moreparticularly non-human stem cells, cord blood stem cells, progenitorcells, bone marrow stem cells, induced pluripotent stem cells,totipotent stem cells or hematopoietic stem cells. Representative humancells are CD34+ cells. Said isolated cell can also be a dendritic cell,killer dendritic cell, a mast cell, a NK-cell, a B-cell or a T-cellselected from the group consisting of inflammatory T-lymphocytes,cytotoxic T-lymphocytes, regulatory T-lymphocytes or helperT-lymphocytes. In another embodiment, said cell can be derived from thegroup consisting of CD4+ T-lymphocytes and CD8+ T-lymphocytes. Prior toexpansion and genetic modification of the cells of the invention, asource of cells can be obtained from a subject through a variety ofnon-limiting methods. Cells can be obtained from a number ofnon-limiting sources, including peripheral blood mononuclear cells, bonemarrow, lymph node tissue, cord blood, thymus tissue, tissue from a siteof infection, ascites, pleural effusion, spleen tissue, and tumors. Incertain embodiments of the present invention, any number of T cell linesavailable and known to those skilled in the art, may be used. In anotherembodiment, said cell can be derived from a healthy donor, from apatient diagnosed with cancer or from a patient diagnosed with aninfection. In another embodiment, said cell is part of a mixedpopulation of cells which present different phenotypic characteristics.In the scope of the present invention is also encompassed a cell lineobtained from a transformed T-cell according to the method previouslydescribed. Modified cells resistant to an immunosuppressive treatmentand susceptible to be obtained by the previous method are encompassed inthe scope of the present invention.

As a preferred embodiment, the present invention provides T-cells or apopulation of T-cells endowed with a CD123 CAR as described above, thatdo not express functional TCR and that a reactive towards CD123 positivecells, for their allogeneic transplantation into patients.

As a more preferred embodiment, the present invention provides T-cellsor a population of T-cells endowed with a CD123 CAR and that a reactivetowards CD123 positive cells as described above, that do not express afunctional TCR and are resistant to a selected drug, for theirallogeneic transplantation into patients treated with said selecteddrug.

In an even more preferred embodiment, the present invention providesT-cells or a population of T-cells endowed with an anti-CD123 CARcomprising a polypeptide of SEQ ID NO. 1+SEQ ID NO. 44, even morepreferably an anti-CD123 CAR comprising 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity with SEQ ID NO. 1+SEQ ID NO. 44, in particular an antiCD123-CAR comprising 85% to 99% identity with SEQ ID NO. 1+SEQ ID NO.44.

Activation and Expansion of T Cells

Whether prior to or after genetic modification of the T cells, even ifthe genetically modified immune cells of the present invention areactivated and proliferate independently of antigen binding mechanisms,the immune cells, particularly T-cells of the present invention can befurther activated and expanded generally using methods as described, forexample, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964;5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869;7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; andU.S. Patent Application Publication No. 20060121005. T cells can beexpanded in vitro or in vivo.

Generally, the T cells of the invention are expanded by contact with anagent that stimulates a CD3 TCR complex and a co-stimulatory molecule onthe surface of the T cells to create an activation signal for theT-cell. For example, chemicals such as calcium ionophore A23187, phorbol12-myristate 13-acetate (PMA), or mitogenic lectins likephytohemagglutinin (PHA) can be used to create an activation signal forthe T-cell.

As non-limiting examples, T cell populations may be stimulated in vitrosuch as by contact with an anti-CD3 antibody, or antigen-bindingfragment thereof, or an anti-CD2 antibody immobilized on a surface, orby contact with a protein kinase C activator (e.g., bryostatin) inconjunction with a calcium ionophore. For co-stimulation of an accessorymolecule on the surface of the T cells, a ligand that binds theaccessory molecule is used. For example, a population of T cells can becontacted with an anti-CD3 antibody and an anti-CD28 antibody, underconditions appropriate for stimulating proliferation of the T cells.Conditions appropriate for T cell culture include an appropriate media(e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 5, (Lonza))that may contain factors necessary for proliferation and viability,including serum (e.g., fetal bovine or human serum), interleukin-2(IL-2), insulin, IFN-g, 1L-4, 1L-7, GM-CSF, -10, -2, 1L-15, TGFp, andTNF- or any other additives for the growth of cells known to the skilledartisan. Other additives for the growth of cells include, but are notlimited to, surfactant, plasmanate, and reducing agents such asN-acetyl-cysteine and 2-mercaptoethanoi. Media can include RPMI 1640,A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1, and X-Vivo 20, Optimizer, withadded amino acids, sodium pyruvate, and vitamins, either serum-free orsupplemented with an appropriate amount of serum (or plasma) or adefined set of hormones, and/or an amount of cytokine(s) sufficient forthe growth and expansion of T cells. Antibiotics, e.g., penicillin andstreptomycin, are included only in experimental cultures, not incultures of cells that are to be infused into a subject. The targetcells are maintained under conditions necessary to support growth, forexample, an appropriate temperature (e.g., 37° C.) and atmosphere (e.g.,air plus 5% C02). T cells that have been exposed to varied stimulationtimes may exhibit different characteristics

In another particular embodiment, said cells can be expanded byco-culturing with tissue or cells. Said cells can also be expanded invivo, for example in the subject's blood after administrating said cellinto the subject.

Therapeutic Applications

In another embodiment, isolated cell obtained by the different methodsor cell line derived from said isolated cell as previously described canbe used as a medicament.

In another embodiment, said medicament can be used for treating cancer,particularly for the treatment of B-cell lymphomas and leukemia in apatient in need thereof.

In another embodiment, said isolated cell according to the invention orcell line derived from said isolated cell can be used in the manufactureof a medicament for treatment of a cancer in a patient in need thereof.

In a particular embodiment, an anti-CD123 CAR expressing T cell isprovided as a medicament for the treatment of AML, of an AML subtype, ofan AML-related complication, of an AML-related condition. In a preferredembodiment, an anti-CD123 CAR expressing T cell wherein said anti-CD123CAR comprises SEQ ID NO 44 is provided as a medicament.

In another embodiment, said medicament can be used for treating aCD123-expressing cell-mediated pathological condition or a conditioncharacterized by the direct or indirect activity of a CD123-expressingcell. In other words, the invention is related to an anti-CD123 CARexpressing T cell comprising 80% to 100% of SEQ ID N044 for its use as amedicament to treat a condition linked to the detrimental activity ofCD123-expressing cells, in particular to treat a condition selected fromAML, AML subtype, AML-related complication, and AML-related conditions;

In another aspect, the present invention relies on methods for treatingpatients in need thereof, said method comprising at least one of thefollowing steps:

-   -   (a) providing an immune-cell obtainable by any one of the        methods previously described;    -   (b) Administrating said transformed immune cells to said        patient,

On one embodiment, said T cells of the invention can undergo robust invivo T cell expansion and can persist for an extended amount of time.

Said treatment can be ameliorating, curative or prophylactic. It may beeither part of an autologous immunotherapy or part of an allogenicimmunotherapy treatment. By autologous, it is meant that cells, cellline or population of cells used for treating patients are originatingfrom said patient or from a Human Leucocyte Antigen (HLA) compatibledonor. By allogeneic is meant that the cells or population of cells usedfor treating patients are not originating from said patient but from adonor.

Cells that can be used with the disclosed methods are described in theprevious section. Said treatment can be used to treat patients diagnosedwherein a pre-malignant or malignant cancer condition characterized byCD123-expressing cells, especially by an overabundance ofCD123-expressing cells. Such conditions are found in hematologiccancers, such as leukemia or malignant lymphoproliferative disorders.Lymphoproliferative disorder can be lymphoma, in particular multiplemyeloma, non-Hodgkin's lymphoma, Burkitt's lymphoma, and follicularlymphoma (small cell and large cell).

Cancers that may be treated may comprise nonsolid tumors (such ashematological tumors, including but not limited to pre-B ALL (pediatricindication), adult ALL, mantle cell lymphoma, diffuse large B-celllymphoma and the like. Types of cancers to be treated with the CARs ofthe invention include, but are not limited leukemia or lymphoidmalignancies. Adult tumors/cancers and pediatric tumors/cancers are alsoincluded.

In one embodiment, the present invention provides a composition for itsuse in the treatment of a CD123 expressing cells-mediated disease, inparticular a CD123 expressing cells—mediated hematologic cancer, saidcomposition comprising said anti-CD123 CAR expressing T cell of theinvention, preferably said anti-CD123 CAR is of SEQ ID NO. 44 or of SEQID NO. 1+SEQ ID NO. 44.

Any other CD123-mediating or CD123-involving malignantlymphoproliferative disorders disclosed herein may be improved with theanti-CD123 CAR-expressing cells of the present invention.

In a preferred embodiment, the cancer that may be treated using theanti-CD123 CAR-expressing cells of the present invention is leukemia, adisease associated to leukemia or a complication thereof.

Leukemias that can be treated using the anti-CD123 CAR-expressing cellsof the present invention can be acute myelogenous leukemia (AML),chronic myelogenous leukemia, melodysplastic syndrome, acute lymphoidleukemia, chronic lymphoid leukemia, and myelodysplastic syndrome.

AML or AML subtypes that may be treated using the anti-CD123CAR-expressing cells of the present invention may be in particular,acute myeloblastic leukemia, minimally differentiated acute myeloblasticleukemia, acute myeloblastic leukemia without maturation, acutemyeloblastic leukemia with granulocytic maturation, promyelocytic oracute promyelocytic leukemia (APL), acute myelomonocytic leukemia,myelomonocytic together with bone marrow eosinophilia, acute monoblasticleukemia (M5a) or acute monocytic leukemia (M5b), acute erythroidleukemias, including erythroleukemia (M6a) and very rare pure erythroidleukemia (M6b), acute megakaryoblastic leukemia, acute basophilicleukemia, acute panmyelosis with myelofibrosis, whether involvingCD123-positive cells.

Subtypes of AML also include, hairy cell leukemia, philadelphiachromosome-positive acute lymphoblastic leukemia.

AML may be classified as AML with specific genetic abnormalities.Classification is based on the ability of karyotype to predict responseto induction therapy, relapse risk, survival.

Accordingly, AML that may be treated using the anti-CD123 CAR-expressingcells of the present invention may be AML with a translocation betweenchromosomes 8 and 21, AML with a translocation or inversion inchromosome 16, AML with a translocation between chromosomes 9 and 11,APL (M3) with a translocation between chromosomes 15 and 17, AML with atranslocation between chromosomes 6 and 9, AML with a translocation orinversion in chromosome 3, AML (megakaryoblastic) with a translocationbetween chromosomes 1 and 22.

The present invention is particularly useful for the treatment of AMLassociated with these particular cytogenetic markers.

The present invention also provides an anti-CD123 CAR expressing T cellfor the treatment of patients with specific cytogenetic subsets of AML,such as patients with t(15;17)(q22;q21) identified using all-transretinoic acid (ATRA)16-19 and for the treatment of patients witht(8;21)(q22;q22) or inv(16)(p13q22)/t(16;16)(p13;q22) identified usingrepetitive doses of high-dose cytarabine.

Preferably, the present invention provides an anti-CD123 CAR expressingT cell for the treatment of patients with aberrations, such as−5/del(5q), −7, abnormalities of 3q, or a complex karyotype, who havebeen shown to have inferior complete remission rates and survival.

Group of Patients

In a preferred embodiment, the invention provides a treatment for AML inpatients over 60 years or in patients of less than 20 years.

In a more preferred embodiment, the present invention provides apediatric treatment, in particular a pediatric treatment against AML, orAML-related diseases or complications.

In still another preferred embodiment, the present invention is used asa treatment in AML patients with low, poor or unfavorable status that isto say with a predicted survival of less than 5 years survival rate. Inthis group, patients suffering AML with the following cytogeneticcharacteristics: −5; 5q; −7; 7q-;11q23; non t(9;11); inv(3); t(3;3);t(6;9); t(9;22) is associated with poor-risk status (Byrd J. C. et al.,Dec. 15, 2002; Blood: 100 (13) and is especially contemplated to betreated according to the present invention or with an object of thepresent invention.

In one embodiment, the anti-CD123 CAR expressing T cell of presentinvention may be used as induction therapy, as post remission therapy ofAML or as a consolidation therapy in patient with AML. Preferably, cellsexpressing at least one anti-CD123 CAR of SEQ ID NO. 1+SEQ ID NO. 44 areused as post remission therapy of AML or as a consolidation therapy inpatient with AML.

In one embodiment, the anti-CD123 CAR expressing T cell of the presentinvention may be used in case of AML relapse, or in case of refractoryor resistant AML. Preferably, cells comprising at least one anti-CD123CAR of SEQ ID NO. 1+SEQ ID NO. 44 of the invention are used in patientswith AML relapse, or with refractory or resistant AML, more preferably,in combination with at least one other anti-cancer drug

In another preferred embodiment, at least one anti-CD123 CAR of SEQ IDNO. 1+SEQ ID NO. 44 expressing cell is used for preventing cancer cellsdevelopment occurring in particular after anti-cancer treatment, duringbone marrow depletion or before bone marrow transplantation, after bonemarrow destruction.

AML Complications

In one particular embodiment the invention provides a medicament thatimproves the health condition of a patient, in particular a patientundergoing a complication related to AML. More preferably, saidengineered anti-CD123 CAR expressing T cell of the invention isexpressing at least one anti-CD123 CAR of SEQ ID NO. 1+SEQ ID NO. 44 andis used as a medicament for the treatment of a complication related toAML.

A complication or disease related to AML may include a precedingmyelodysplasia phase, secondary leukemia, in particular secondary AML,high white blood cell count, and absence of Auer rods. Among others,leukostasis and involvement of the central nervous system (CNS),Hyperleukocytosis, residual disease, are also considered as acomplication or disease related to AML.

AML Associated Diseases

In one embodiment, the present invention also provides an anti-CD123 CARexpressing T cell for the treatment of a pathological condition relatedto AML. Preferably, the present invention provides a cell expressing atleast one anti-CD123 CAR of SEQ ID NO. 1+SEQ ID NO. 44 for the treatmentof a pathological condition related to AML. The present inventionprovides a therapy for AML related myeloid neoplasms, for acute myeloidleukemia and myelodysplastic syndrome, a treatment of relapsed orrefractory acute myeloid leukemia, a treatment of relapsed or refractoryacute promyelocytic leukemia in adults, a treatment for acute promyeloidleukaemia, a treatment of acute myeloid leukemia in adults over 60years.

According to another aspect, the present invention provides acomposition for the treatment of AML associated diseases, in particularhematologic malignancy related to AML.

Hematologic malignancy related to AML conditions include myelodysplasiasyndromes (MDS, formerly known as “preleukemia”) which are a diversecollection of hematological conditions united by ineffective production(or dysplasia) of myeloid blood cells and risk of transformation to AM

In another embodiment, the invention provides a medicament that improvesthe health state of a patient suffering multiple myeloma.

Other pathological conditions or genetic syndromes associated with therisk of AML can be improved with the adequate use of the presentinvention, said genetic syndromes include Down syndrome, trisomy,Fanconi anemia, Bloom syndrome, Ataxia-telangiectasia, Diamond-Blackfananemia, Schwachman-Diamond syndrome, Li-Fraumeni syndrome,Neurofibromatosis type 1, Severe congenital neutropenia (also calledKostmann syndrome)

Other CD123-Mediated Pathological Conditions

According to another aspect, the present invention provides acomposition for the treatment of CD123+cell-mediated diseases. TheseCD123+cell mediated diseases include inflammation, autoimmune diseases.

In particular, the present invention can be used for the treatment ofCD123+cell mediated diseases such as inflammation of thegastrointestinal mucosae and more particularly, inflammatory boweldiseases, nasal allergy, inflammation of the skin such as juveniledermatomyositis, hematodermia.

The present invention can be used as a medicament for the treatment ofCD123+cell mediated diseases such as autoimmune diseases in particularKikushi disease.

Preferably, the present invention provides a treatment for a recurrentinfection including infection due to viruses such as Epstein-Barr virus,herpes simplex virus, in particular oncogenic viruses, HHV-8, HHV-6,HTLV or HIV, parasitic infection such as infection due to plasmodiumfalciparum, Plasmodium vivax, Plasmodium ovale, or Plasmodium malariae.

In particular, the present invention provides a treatment forEpstein-Barr virus lymphadenitis, herpes simplex virus lymphadenitis.

In another aspect, the present invention provides a composition for thetreatment of systemic lupus erythematosus lymphadenitis, tuberculosis,cystic fibrosis, hepatitis, biliary atresia, in particular virus-inducedhepatitis or biliary atresia in children, autoimmune hepatitis; primarybiliary cirrhosis.

Composition Comprising an Engineered T Cells According to the Inventionfor Use as a Medicament and Method

The present invention also provides a composition for its use or amethod for treating a disease. In one aspect, the disease is ahematologic cancer, in particular a stem cell cancer including but isnot limited to leukemia (such as acute myelogenous leukemia (AML),chronic myelogenous leukemia, acute lymphoid leukemia, chronic lymphoidleukemia and myelodysplasia syndrome) and malignant lymphoproliferativeconditions, including lymphoma (such as multiple myeloma, non-Hodgkin'slymphoma, Burkitt's lymphoma, and small cell- and large cell-follicularlymphoma), or a complication thereof.

The present invention also provides a composition for its use or amethod for inhibiting the proliferation or reducing a CD123-expressingcell population or activity in a patient. An exemplary method includescontacting a population of cells comprising a CD123-expressing cell witha CD 123 CART cell of the invention that binds to the CD123-expressingcell.

In a more specific aspect, the present invention provides a compositionfor its use or a method for inhibiting the proliferation or reducing thepopulation of cancer cells expressing CD 123 in a patient, the methodscomprising contacting the CD123-expressing cancer cell population with aCD 123 CART cell of the invention that binds to the CD123-expressingcell, binding of a CD 123 CART cell of the invention to theCD123-expressing cancer cell resulting in the destruction of theCD123-expressing cancer cells

In certain aspects, the CD 123 CART cell of the invention reduces thequantity, number, amount or percentage of cells and/or cancer cells byat least 25%, at least 30%, at least 40%, at least 50%, at least 65%, atleast 75%, at least 85%, at least 95%, or at least 99% (to undetectablelevel) in a subject with or animal model for myeloid leukemia or anothercancer associated with CD123-expressing cells, relative to a negativecontrol.

The present invention also provides a composition for its use or amethod for preventing, treating and/or managing a disorder or conditionassociated with CD123-expressing cells (e.g., associated with ahematologic cancer), the methods comprising administering to a subjectin need a CD 123 CART cell of the invention that binds to theCD123-expressing cell. In one aspect, the subject is a human.Non-limiting examples of disorders associated with CD123-expressingcells include autoimmune disorders (such as lupus), inflammatorydisorders (such as allergies, IBD, and asthma) and cancers (such ashematological cancers, in particular AML or AML complications).

The present invention also provides a composition for its use or amethod for preventing, treating and/or managing a disease associatedwith CD123-expressing cells, the method comprising administering to asubject in need a CD 123 CART cell of the invention that binds to theCD123-expressing cell. In one aspect, the subject is a human.Non-limiting examples of diseases associated with CD123-expressing cellsinclude Acute Myeloid Leukemia (AML), myelodysplasia, B-cell AcuteLymphoid Leukemia, T-cell Acute Lymphoid Leukemia, hairy cell leukemia,blastic plasmacytoid dendritic cell neoplasm, chronic myeloid leukemia,hodgkin lymphoma.

The present invention provides a composition for its use or a method fortreating or preventing relapse of cancer associated withCD123-expressing cells, the method comprising administering to a subjectin need thereof a CD 123 CART cell of the invention that binds to the CD123-expressing cell. In another aspect, the methods compriseadministering to the subject in need thereof an effective amount of a CD123 CART cell of the invention that binds to the CD123-expressing cellin combination with an effective amount of another therapy.

In one aspect, CD 123 is considered to be a “cancer stem cell” marker inAML. Therefore, a CD 123 CART cell of the invention can prevent relapseof AML, or even treat AML that is mostly CD 123-negative but with a“stem” population of CD 123+ cells (a CD123-expressing cells).

In one aspect, the invention provides compositions and methods fortreating subjects that have undergone treatment for a disease ordisorder associated with elevated expression levels of CD 19, andexhibits a disease or disorder associated with elevated levels of CD123.

In one aspect, B-cell acute lymphoid leukemia (ALL) is an example ofdisease requiring a serial treatment using CART cells. For example,treatment with anti-CD 19 CAR T cells can sometimes result inCD19-negative relapse, which can be treated with anti-CD123 CAR T cellsof the invention. Alternatively, the present invention includes dualtargeting of B-ALL using CART cells comprising an anti-CD 19 CAR and ananti-CD 123 CAR.

The treatment with the engineered immune cells according to theinvention may be in combination with one or more therapies againstcancer selected from the group of antibodies therapy, chemotherapy,cytokines therapy, dendritic cell therapy, gene therapy, hormonetherapy, laser light therapy and radiation therapy.

Preferably, the treatment with the engineered immune cells according tothe invention may be administered in combination (e.g., before,simultaneously or following) with one or more therapies against cancerselected from Aracytine, Cytosine Arabinoside, amsacrine, Daunorubicine,Idarubicine, Novantrone, Mitoxantrone, Vepeside, Etoposide (VP16),arsenic trioxyde, transretinoic acid, combination of arsenic trioxyde,transretinoic acid, mechlorethamine, procarbazine, chlorambucil, andcombination thereof.

According to a preferred embodiment of the invention, said treatment canbe administrated into patients undergoing an immunosuppressivetreatment. Indeed, the present invention preferably relies on cells orpopulation of cells, which have been made resistant to at least oneimmunosuppressive agent due to the inactivation of a gene encoding areceptor for such immunosuppressive agent. In this aspect, theimmunosuppressive treatment should help the selection and expansion ofthe T-cells according to the invention within the patient.

The administration of the cells or population of cells according to thepresent invention may be carried out in any convenient manner, includingby aerosol inhalation, injection, ingestion, transfusion, implantationor transplantation. The compositions described herein may beadministered to a patient subcutaneously, intradermaly, intratumorally,intranodally, intramedullary, intramuscularly, by intravenous orintralymphatic injection, or intraperitoneally. In one embodiment, thecell compositions of the present invention are preferably administeredby intravenous injection.

The administration of the cells or population of cells can consist ofthe administration of 10⁴-10⁹ cells per kg body weight, preferably 10⁵to 10⁶ cells/kg body weight including all integer values of cell numberswithin those ranges. The cells or population of cells can beadministrated in one or more doses. In another embodiment, saideffective amount of cells are administrated as a single dose. In anotherembodiment, said effective amount of cells are administrated as morethan one dose over a period time. Timing of administration is within thejudgment of managing physician and depends on the clinical condition ofthe patient. The cells or population of cells may be obtained from anysource, such as a blood bank or a donor. While individual needs vary,determination of optimal ranges of effective amounts of a given celltype for a particular disease or conditions within the skill of the art.An effective amount means an amount which provides a therapeutic orprophylactic benefit. The dosage administrated will be dependent uponthe age, health and weight of the recipient, kind of concurrenttreatment, if any, frequency of treatment and the nature of the effectdesired.

In another embodiment, said effective amount of cells or compositioncomprising those cells are administrated parenterally. Saidadministration can be an intravenous administration. Said administrationcan be directly done by injection within a tumor.

In certain embodiments of the present invention, cells are administeredto a patient in conjunction with (e.g., before, simultaneously orfollowing) any number of relevant treatment modalities, including butnot limited to treatment with agents such as antiviral therapy,cidofovir and interleukin-2, Cytarabine (also known as ARA-C) ornatalizimab treatment for MS patients or efaliztimab treatment forpsoriasis patients or other treatments for PML patients. In furtherembodiments, the T cells of the invention may be used in combinationwith chemotherapy, radiation, immunosuppressive agents, such ascyclosporin, azathioprine, methotrexate, mycophenolate, and FK506,antibodies, or other immunoablative agents such as CAMPATH, anti-CD3antibodies or other antibody therapies, cytoxin, fludaribine,cyclosporin, FK506, rapamycin, mycoplienolic acid, steroids, FR901228,cytokines, and irradiation. These drugs inhibit either the calciumdependent phosphatase calcineurin (cyclosporine and FK506) or inhibitthe p70S6 kinase that is important for growth factor induced signaling(rapamycin) (Henderson, Naya et al. 1991; Liu, Albers et al. 1992;Bierer, Hollander et al. 1993). In a further embodiment, the cellcompositions of the present invention are administered to a patient inconjunction with (e.g., before, simultaneously or following) bone marrowtransplantation, T cell ablative therapy using either chemotherapyagents such as, fludarabine, external-beam radiation therapy (XRT),cyclophosphamide, or antibodies such as OKT3 or CAMPATH, In anotherembodiment, the cell compositions of the present invention areadministered following B-cell ablative therapy such as agents that reactwith CD20, e.g., Rituxan. For example, in one embodiment, subjects mayundergo standard treatment with high dose chemotherapy followed byperipheral blood stem cell transplantation. In certain embodiments,following the transplant, subjects receive an infusion of the expandedimmune cells of the present invention. In an additional embodiment,expanded cells are administered before or following surgery.

In certain embodiments of the present invention, anti-CD123 CARexpressing cells are administered to a patient in conjunction (e.g.,before, simultaneously or following) with a drug selected fromAracytine, Cytosine Arabinoside, amsacrine, Daunorubicine, Idarubicine,Novantrone, Mitoxantrone, Vepeside, Etoposide (VP16), arsenic trioxyde,transretinoic acid, mechlorethamine, procarbazine, chlorambucil, andcombination thereof. In these embodiments anti-CD123 CAR expressingcells may be resistant to the particular drug or combination of drugsthat is (are) administered in conjunction with anti-CD123 CAR expressingcells.

In other embodiments of the present invention, anti-CD123 CAR expressingcells are administered to a patient in conjunction with a drug selectedfrom cytarabine, anthracyclines, 6-thioguanine, hydroxyurea, prednisone,and combination thereof.

Other Definitions

-   -   Unless otherwise specified, “a,” “an,” “the,” and “at least one”        are used interchangeably and mean one or more than one. Amino        acid residues in a polypeptide sequence are designated herein        according to the one-letter code, in which, for example, Q means        Gln or Glutamine residue, R means Arg or Arginine residue and D        means Asp or Aspartic acid residue.    -   Amino acid substitution means the replacement of one amino acid        residue with another, for instance the replacement of an        Arginine residue with a Glutamine residue in a peptide sequence        is an amino acid substitution.    -   Nucleotides are designated as follows: one-letter code is used        for designating the base of a nucleoside: an is adenine, t is        thymine, c is cytosine, and g is guanine. For the degenerated        nucleotides, r represents g or a (purine nucleotides), k        represents g or t, s represents g or c, w represents a or t, m        represents a or c, y represents t or c (pyrimidine nucleotides),        d represents g, a or t, v represents g, a or c, b represents g,        t or c, h represents a, t or c, and n represents g, a, t or c.    -   “As used herein, “nucleic acid” or “polynucleotides” refers to        nucleotides and/or polynucleotides, such as deoxyribonucleic        acid (DNA) or ribonucleic acid (RNA), oligonucleotides,        fragments generated by the polymerase chain reaction (PCR), and        fragments generated by any of ligation, scission, endonuclease        action, and exonuclease action. Nucleic acid molecules can be        composed of monomers that are naturally-occurring nucleotides        (such as DNA and RNA), or analogs of naturally-occurring        nucleotides (e.g., enantiomeric forms of naturally-occurring        nucleotides), or a combination of both. Modified nucleotides can        have alterations in sugar moieties and/or in pyrimidine or        purine base moieties. Sugar modifications include, for example,        replacement of one or more hydroxyl groups with halogens, alkyl        groups, amines, and azido groups, or sugars can be        functionalized as ethers or esters. Moreover, the entire sugar        moiety can be replaced with sterically and electronically        similar structures, such as aza-sugars and carbocyclic sugar        analogs. Examples of modifications in a base moiety include        alkylated purines and pyrimidines, acylated purines or        pyrimidines, or other well-known heterocyclic substitutes.        Nucleic acid monomers can be linked by phosphodiester bonds or        analogs of such linkages. Nucleic acids can be either single        stranded or double stranded.    -   By chimeric antigen receptor (CAR) is intended molecules that        combine a binding domain against a component present on the        target cell, for example an antibody-based specificity for a        desired antigen (e.g., tumor antigen) with a T cell        receptor-activating intracellular domain to generate a chimeric        protein that exhibits a specific anti-target cellular immune        activity. Generally, CAR consists of an extracellular single        chain antibody (scFv Fc) fused to the intracellular signaling        domain of the T cell antigen receptor complex zeta chain (scFv        Fc:ζ) and have the ability, when expressed in T cells, to        redirect antigen recognition based on the monoclonal antibody's        specificity. One example of CAR used in the present invention is        a CAR directing against CD123 antigen and can comprise as non        limiting example the amino acid sequences: SEQ ID NO: 23 to 48.    -   The term “endonuclease” refers to any wild-type or variant        enzyme capable of catalyzing the hydrolysis (cleavage) of bonds        between nucleic acids within a DNA or RNA molecule, preferably a        DNA molecule. Endonucleases do not cleave the DNA or RNA        molecule irrespective of its sequence, but recognize and cleave        the DNA or RNA molecule at specific polynucleotide sequences,        further referred to as “target sequences” or “target sites”.        Endonucleases can be classified as rare-cutting endonucleases        when having typically a polynucleotide recognition site greater        than 12 base pairs (bp) in length, more preferably of 14-55 bp.        Rare-cutting endonucleases significantly increase HR by inducing        DNA double-strand breaks (DSBs) at a defined locus (Perrin,        Buckle et al. 1993; Rouet, Smih et al. 1994; Choulika, Perrin et        al. 1995; Pingoud and Silva 2007). Rare-cutting endonucleases        can for example be a homing endonuclease (Paques and Duchateau        2007), a chimeric Zinc-Finger nuclease (ZFN) resulting from the        fusion of engineered zinc-finger domains with the catalytic        domain of a restriction enzyme such as Fokl (Porteus and Carroll        2005), a Cas9 endonuclease from CRISPR system (Gasiunas,        Barrangou et al. 2012; Jinek, Chylinski et al. 2012; Cong, Ran        et al. 2013; Mali, Yang et al. 2013) or a chemical endonuclease        (Eisenschmidt, Lanio et al. 2005; Arimondo, Thomas et al. 2006).        In chemical endonucleases, a chemical or peptidic cleaver is        conjugated either to a polymer of nucleic acids or to another        DNA recognizing a specific target sequence, thereby targeting        the cleavage activity to a specific sequence. Chemical        endonucleases also encompass synthetic nucleases like conjugates        of orthophenanthroline, a DNA cleaving molecule, and        triplex-forming oligonucleotides (TFOs), known to bind specific        DNA sequences (Kalish and Glazer 2005). Such chemical        endonucleases are comprised in the term “endonuclease” according        to the present invention.    -   By a “TALE-nuclease” (TALEN) is intended a fusion protein        consisting of a nucleic acid-binding domain typically derived        from a Transcription Activator Like Effector (TALE) and one        nuclease catalytic domain to cleave a nucleic acid target        sequence. The catalytic domain is preferably a nuclease domain        and more preferably a domain having endonuclease activity, like        for instance I-Tevl, ColE7, NucA and Fok-I. In a particular        embodiment, the TALE domain can be fused to a meganuclease like        for instance I-Crel and I-Onul or functional variant thereof. In        a more preferred embodiment, said nuclease is a monomeric        TALE-Nuclease. A monomeric TALE-Nuclease is a TALE-Nuclease that        does not require dimerization for specific recognition and        cleavage, such as the fusions of engineered TAL repeats with the        catalytic domain of I-Tevl described in WO2012138927.        Transcription Activator like Effector (TALE) are proteins from        the bacterial species Xanthomonas comprise a plurality of        repeated sequences, each repeat comprising di-residues in        position 12 and 13 (RVD) that are specific to each nucleotide        base of the nucleic acid targeted sequence. Binding domains with        similar modular base-per-base nucleic acid binding properties        (MBBBD) can also be derived from new modular proteins recently        discovered by the applicant in a different bacterial species.        The new modular proteins have the advantage of displaying more        sequence variability than TAL repeats. Preferably, RVDs        associated with recognition of the different nucleotides are HD        for recognizing C, NG for recognizing T, NI for recognizing A,        NN for recognizing G or A, NS for recognizing A, C, G or T, HG        for recognizing T, IG for recognizing T, NK for recognizing G,        HA for recognizing C, ND for recognizing C, HI for recognizing        C, HN for recognizing G, NA for recognizing G, SN for        recognizing G or A and YG for recognizing T, TL for recognizing        A, VT for recognizing A or G and SW for recognizing A. In        another embodiment, critical amino acids 12 and 13 can be        mutated towards other amino acid residues in order to modulate        their specificity towards nucleotides A, T, C and G and in        particular to enhance this specificity. TALE-nuclease have been        already described and used to stimulate gene targeting and gene        modifications (Boch, Scholze et al. 2009; Moscou and Bogdanove        2009; Christian, Cermak et al. 2010; Li, Huang et al. 2011).        Engineered TAL-nucleases are commercially available under the        trade name TALEN™ (Cellectis, 8 rue de la Croix Jarry, 75013        Paris, France).

The rare-cutting endonuclease according to the present invention canalso be a Cas9 endonuclease. Recently, a new genome engineering tool hasbeen developed based on the RNA-guided Cas9 nuclease (Gasiunas,Barrangou et al. 2012; Jinek, Chylinski et al. 2012; Cong, Ran et al.2013; Mali, Yang et al. 2013) from the type II prokaryotic CRISPR(Clustered Regularly Interspaced Short palindromic Repeats) adaptiveimmune system (see for review (Sorek, Lawrence et al. 2013)). The CRISPRAssociated (Cas) system was first discovered in bacteria and functionsas a defense against foreign DNA, either viral or plasmid.CRISPR-mediated genome engineering first proceeds by the selection oftarget sequence often flanked by a short sequence motif, referred as theproto-spacer adjacent motif (PAM). Following target sequence selection,a specific crRNA, complementary to this target sequence is engineered.Trans-activating crRNA (tracrRNA) required in the CRISPR type II systemspaired to the crRNA and bound to the provided Cas9 protein. Cas9 acts asa molecular anchor facilitating the base pairing of tracRNA with cRNA(Deltcheva, Chylinski et al. 2011). In this ternary complex, the dualtracrRNA:crRNA structure acts as guide RNA that directs the endonucleaseCas9 to the cognate target sequence. Target recognition by theCas9-tracrRNA:crRNA complex is initiated by scanning the target sequencefor homology between the target sequence and the crRNA. In addition tothe target sequence-crRNA complementarity, DNA targeting requires thepresence of a short motif adjacent to the protospacer (protospaceradjacent motif—PAM). Following pairing between the dual-RNA and thetarget sequence, Cas9 subsequently introduces a blunt double strandbreak 3 bases upstream of the PAM motif (Garneau, Dupuis et al. 2010).

Rare-cutting endonuclease can be a homing endonuclease, also known underthe name of meganuclease. Such homing endonucleases are well-known tothe art (Stoddard 2005). Homing endonucleases recognize a DNA targetsequence and generate a single- or double-strand break. Homingendonucleases are highly specific, recognizing DNA target sites rangingfrom 12 to 45 base pairs (bp) in length, usually ranging from 14 to 40bp in length. The homing endonuclease according to the invention may forexample correspond to a LAGLIDADG endonuclease, to a HNH endonuclease,or to a GIY-YIG endonuclease. Preferred homing endonuclease according tothe present invention can be an I-Crel variant.

-   -   By “delivery vector” or “delivery vectors” is intended any        delivery vector which can be used in the present invention to        put into cell contact (i.e “contacting”) or deliver inside cells        or subcellular compartments (i.e “introducing”) agents/chemicals        and molecules (proteins or nucleic acids) needed in the present        invention. It includes, but is not limited to liposomal delivery        vectors, viral delivery vectors, drug delivery vectors, chemical        carriers, polymeric carriers, lipoplexes, polyplexes,        dendrimers, microbubbles (ultrasound contrast agents),        nanoparticles, emulsions or other appropriate transfer vectors.        These delivery vectors allow delivery of molecules, chemicals,        macromolecules (genes, proteins), or other vectors such as        plasmids, peptides developed by Diatos. In these cases, delivery        vectors are molecule carriers. By “delivery vector” or “delivery        vectors” is also intended delivery methods to perform        transfection.    -   The terms “vector” or “vectors” refer to a nucleic acid molecule        capable of transporting another nucleic acid to which it has        been linked. A “vector” in the present invention includes, but        is not limited to, a viral vector, a plasmid, a RNA vector or a        linear or circular DNA or RNA molecule which may consists of a        chromosomal, non chromosomal, semi-synthetic or synthetic        nucleic acids. Preferred vectors are those capable of autonomous        replication (episomal vector) and/or expression of nucleic acids        to which they are linked (expression vectors). Large numbers of        suitable vectors are known to those of skill in the art and        commercially available.

Viral vectors include retrovirus, adenovirus, parvovirus (e. g.adenoassociated viruses), coronavirus, negative strand RNA viruses suchas orthomyxovirus (e. g., influenza virus), rhabdovirus (e. g., rabiesand vesicular stomatitis virus), paramyxovirus (e. g. measles andSendai), positive strand RNA viruses such as picornavirus andalphavirus, and double-stranded DNA viruses including adenovirus,herpesvirus (e. g., Herpes Simplex virus types 1 and 2, Epstein-Barrvirus, cytomegalovirus), and poxvirus (e. g., vaccinia, fowlpox andcanarypox). Other viruses include Norwalk virus, togavirus, flavivirus,reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example.Examples of retroviruses include: avian leukosis-sarcoma, mammalianC-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus,spumavirus (Coffin, J. M., Retroviridae: The viruses and theirreplication, In Fundamental Virology, Third Edition, B. N. Fields, etal., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

-   -   By “lentiviral vector” is meant HIV-Based lentiviral vectors        that are very promising for gene delivery because of their        relatively large packaging capacity, reduced immunogenicity and        their ability to stably transduce with high efficiency a large        range of different cell types. Lentiviral vectors are usually        generated following transient transfection of three (packaging,        envelope and transfer) or more plasmids into producer cells.        Like HIV, lentiviral vectors enter the target cell through the        interaction of viral surface glycoproteins with receptors on the        cell surface. On entry, the viral RNA undergoes reverse        transcription, which is mediated by the viral reverse        transcriptase complex. The product of reverse transcription is a        double-stranded linear viral DNA, which is the substrate for        viral integration in the DNA of infected cells. By “integrative        lentiviral vectors (or LV)”, is meant such vectors as        nonlimiting example, that are able to integrate the genome of a        target cell. At the opposite by “non-integrative lentiviral        vectors (or NILV)” is meant efficient gene delivery vectors that        do not integrate the genome of a target cell through the action        of the virus integrase.    -   Delivery vectors and vectors can be associated or combined with        any cellular permeabilization techniques such as sonoporation or        electroporation or derivatives of these techniques.    -   By cell or cells is intended any eukaryotic living cells,        primary cells and cell lines derived from these organisms for in        vitro cultures.    -   By “primary cell” or “primary cells” are intended cells taken        directly from living tissue (i.e. biopsy material) and        established for growth in vitro, that have undergone very few        population doublings and are therefore more representative of        the main functional components and characteristics of tissues        from which they are derived from, in comparison to continuous        tumorigenic or artificially immortalized cell lines.

As non-limiting examples cell lines can be selected from the groupconsisting of CHO-K1 cells; HEK293 cells; Caco2 cells; U2-OS cells; NIH3T3 cells; NSO cells; SP2 cells; CHO-S cells; DG44 cells; K-562 cells,U-937 cells; MRC5 cells; IMR90 cells; Jurkat cells; HepG2 cells; HeLacells; HT-1080 cells; HCT-116 cells; Hu-h7 cells; Huvec cells; Molt 4cells.

All these cell lines can be modified by the method of the presentinvention to provide cell line models to produce, express, quantify,detect, study a gene or a protein of interest; these models can also beused to screen biologically active molecules of interest in research andproduction and various fields such as chemical, biofuels, therapeuticsand agronomy as non-limiting examples.

-   -   by “mutation” is intended the substitution, deletion, insertion        of up to one, two, three, four, five, six, seven, eight, nine,        ten, eleven, twelve, thirteen, fourteen, fifteen, twenty, twenty        five, thirty, fourty, fifty, or more nucleotides/amino acids in        a polynucleotide (cDNA, gene) or a polypeptide sequence. The        mutation can affect the coding sequence of a gene or its        regulatory sequence. It may also affect the structure of the        genomic sequence or the structure/stability of the encoded mRNA.    -   by “variant(s)”, it is intended a repeat variant, a variant, a        DNA binding variant, a TALE-nuclease variant, a polypeptide        variant obtained by mutation or replacement of at least one        residue in the amino acid sequence of the parent molecule.    -   by “functional variant” is intended a catalytically active        mutant of a protein or a protein domain; such mutant may have        the same activity compared to its parent protein or protein        domain or additional properties, or higher or lower activity.    -   “identity” refers to sequence identity between two nucleic acid        molecules or polypeptides. Identity can be determined by        comparing a position in each sequence which may be aligned for        purposes of comparison. When a position in the compared sequence        is occupied by the same base, then the molecules are identical        at that position. A degree of similarity or identity between        nucleic acid or amino acid sequences is a function of the number        of identical or matching nucleotides at positions shared by the        nucleic acid sequences. Various alignment algorithms and/or        programs may be used to calculate the identity between two        sequences, including FASTA, or BLAST which are available as a        part of the GCG sequence analysis package (University of        Wisconsin, Madison, Wis.), and can be used with, e.g., default        setting. For example, polypeptides having at least 70%, 85%,        90%, 95%, 98% or 99% identity to specific polypeptides described        herein and preferably exhibiting substantially the same        functions, as well as polynucleotide encoding such polypeptides,        are contemplated.    -   “similarity” describes the relationship between the amino acid        sequences of two or more polypeptides. BLASTP may also be used        to identify an amino acid sequence having at least 70%, 75%,        80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, 99% sequence        similarity to a reference amino acid sequence using a similarity        matrix such as BLOSUM45, BLOSUM62 or BLOSUM80. Unless otherwise        indicated a similarity score will be based on use of BLOSUM62.        When BLASTP is used, the percent similarity is based on the        BLASTP positives score and the percent sequence identity is        based on the BLASTP identities score. BLASTP “Identities” shows        the number and fraction of total residues in the high scoring        sequence pairs which are identical; and BLASTP “Positives” shows        the number and fraction of residues for which the alignment        scores have positive values and which are similar to each other.        Amino acid sequences having these degrees of identity or        similarity or any intermediate degree of identity of similarity        to the amino acid sequences disclosed herein are contemplated        and encompassed by this disclosure. The polynucleotide sequences        of similar polypeptides are deduced using the genetic code and        may be obtained by conventional means. For example, a functional        variant of pTalpha can have 70%, 75%, 80%, 85%, 87.5%, 90%,        92.5%, 95%, 97.5%, 98%, 99% sequence similarity to the amino        acid sequence of SEQ ID NO: 107. A polynucleotide encoding such        a functional variant would be produced by reverse translating        its amino acid sequence using the genetic code.    -   “signal-transducing domain” or “co-stimulatory ligand” refers to        a molecule on an antigen presenting cell that specifically binds        a cognate co-stimulatory molecule on a T-cell, thereby providing        a signal which, in addition to the primary signal provided by,        for instance, binding of a TCR/CD3 complex with an MHC molecule        loaded with peptide, mediates a T cell response, including, but        not limited to, proliferation activation, differentiation and        the like. A co-stimulatory ligand can include but is not limited        to CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L,        inducible costimulatory ligand (ICOS-L), intercellular adhesion        molecule (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB,        HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, an agonist        or antibody that binds Toll ligand receptor and a ligand that        specifically binds with B7-H3. A co-stimulatory ligand also        encompasses, inter alia, an antibody that specifically binds        with a co-stimulatory molecule present on a T cell, such as but        not limited to, CD27, CD28, 4-IBB, OX40, CD30, CD40, PD-1, ICOS,        lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7,        LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83.

A “co-stimulatory molecule” refers to the cognate binding partner on a Tcell that specifically binds with a co-stimulatory ligand, therebymediating a co-stimulatory response by the cell, such as, but notlimited to proliferation. Co-stimulatory molecules include, but are notlimited to an MHC class I molecule, BTLA and Toll ligand receptor.

A “co-stimulatory signal” as used herein refers to a signal, which incombination with primary signal, such as TCR/CD3 ligation, leads to Tcell proliferation and/or upregulation or downregulation of keymolecules.

The term “extracellular ligand-binding domain” as used herein is definedas an oligo- or polypeptide that is capable of binding a ligand.Preferably, the domain will be capable of interacting with a cellsurface molecule. For example, the extracellular ligand-binding domainmay be chosen to recognize a ligand that acts as a cell surface markeron target cells associated with a particular disease state. Thusexamples of cell surface markers that may act as ligands include thoseassociated with viral, bacterial and parasitic infections, autoimmunedisease and cancer cells.

The term “subject” or “patient” as used herein includes all members ofthe animal kingdom including non-human primates and humans.

The term “relapsed” refers to a situation where a subject or a mammal,who has had a remission of cancer after therapy has a return of cancercells.

The term “refractory or resistant” refers to a circumstance where asubject or a mammal, even after intensive treatment, has residual cancercells in his body.

The term “drug resistance” refers to the condition when a disease doesnot respond to the treatment of a drug or drugs. Drug resistance can beeither intrinsic (or primary resistance), which means the disease hasnever been responsive to the drug or drugs, or it can be acquired, whichmeans the disease ceases responding to a drug or drugs that the diseasehad previously responded to (secondary resistance). In certainembodiments, drug resistance is intrinsic. In certain embodiments, thedrug resistance is acquired.

The term “hematologic malignancy” or “hematologic cancer” refers to acancer of the body's blood-bone marrow and/or lymphatic tissue. Examplesof hematological malignancies include, for instance, myelodysplasia,leukemia, lymphomas, such as cutaneous Lymphomas, non-Hodgkin'slymphoma, Hodgkin's disease (also called Hodgkin's lymphoma), andmyeloma, such as acute lymphocytic leukemia (ALL), acute myeloidleukemia (AML), acute promyelocytic leukemia (APL), chronic lymphocyticleukemia (CLL), chronic myeloid leukemia (CML), chronic neutrophilicleukemia (CNL), acute undifferentiated leukemia (AUL), anaplasticlarge-cell lymphoma (ALCL), prolymphocytic leukemia (PML), juvenilemyelomonocyctic leukemia (JMML), adult T-cell ALL, AML with trilineagemyelodysplasia (AML/TMDS), mixed lineage leukemia (MLL), myelodysplasticsyndromes (MDSs), myeloproliferative disorders (MPD), and multiplemyeloma (MM).

The term “leukemia” refers to malignant neoplasms of the blood-formingtissues, including, but not limited to, chronic lymphocytic leukemia orchronic lymphoid leukemia, chronic myelocytic leukemia, or chronicmyelogenous leukemia, acute lymphoblastic leukemia, acute myeloidleukemia or acute myelogenous leukemia (AML) and acute myeloblasticleukemia.

The above written description of the invention provides a manner andprocess of making and using it such that any person skilled in this artis enabled to make and use the same, this enablement being provided inparticular for the subject matter of the appended claims, which make upa part of the original description.

Where a numerical limit or range is stated herein, the endpoints areincluded. Also, all values and subranges within a numerical limit orrange are specifically included as if explicitly written out.

The above description is presented to enable a person skilled in the artto make and use the invention, and is provided in the context of aparticular application and its requirements. Various modifications tothe preferred embodiments will be readily apparent to those skilled inthe art, and the generic principles defined herein may be applied toother embodiments and applications without departing from the spirit andscope of the invention. Thus, this invention is not intended to belimited to the embodiments shown, but is to be accorded the widest scopeconsistent with the principles and features disclosed herein.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples, which areprovided herein for purposes of illustration only, and are not intendedto be limiting unless otherwise specified.

General Method

Primary T-Cell Cultures

T cells were purified from Buffy coat samples provided by EFS(Etablissement Francais du Sang, Paris, France) using Ficoll gradientdensity medium. The PBMC layer was recovered and T cells were purifiedusing a commercially available T-cell enrichment kit. Purified T cellswere activated in X-Vivo™-15 medium (Lonza) supplemented with 20 ng/mLHuman IL-2, 5% Human, and Dynabeads Human T activator CD3/CD28 at abead:cell ratio 1:1 (Life Technologies).

CAR mRNA Transfection

Transfections were done at Day 4 or Day 11 after T-cell purification andactivation. 5 millions of cells were transfected with 15 μg of mRNAencoding the different CAR constructs. CAR mRNAs were produced using T7mRNA polymerase transfections done using Cytopulse technology, byapplying two 0.1 mS pulses at 3000V/cm followed by four 0.2 mS pulses at325V/cm in 0.4 cm gap cuvettes in a final volume of 200 μl of“Cytoporation buffer T” (BTX Harvard Apparatus). Cells were immediatelydiluted in X-Vivo™-15 media and incubated at 37° C. with 5% CO₂. IL-2was added 2 h after electroporation at 20 ng/mL.

Degranulation Assay (CD107a Mobilization)

T-cells were incubated in 96-well plates (40,000 cells/well), togetherwith an equal amount of cells expressing various levels of the CD123protein. Co-cultures were maintained in a final volume of 100 μl ofX-Vivo™-15 medium (Lonza) for 6 hours at 37° C. with 5% CO₂. CD107astaining was done during cell stimulation, by the addition of afluorescent anti-CD107a antibody at the beginning of the co-culture,together with 1 μg/ml of anti-CD49d, 1 μg/ml of anti-CD28, and 1×Monensin solution. After the 6 h incubation period, cells were stainedwith a fixable viability dye and fluorochrome-conjugated anti-CD8 andanalyzed by flow cytometry. The degranulation activity was determined asthe % of CD8+/CD107a+ cells, and by determining the mean fluorescenceintensity signal (MFI) for CD107a staining among CD8+ cells.Degranulation assays were carried out 24 h after mRNA transfection.

IFN Gamma Release Assay

T-cells were incubated in 96-well plates (40,000 cells/well), togetherwith cell lines expressing various levels of the CD123 protein.Co-cultures were maintained in a final volume of 100 μl of X-Vivo™-15medium (Lonza) for 24 hours at 37° C. with 5% CO₂. After this incubationperiod the plates were centrifuged at 1500 rpm for 5 minutes and thesupernatants were recovered in a new plate. IFN gamma detection in thecell culture supernatants was done by ELISA assay. The IFN gamma releaseassays were carried by starting the cell co-cultures 24 h after mRNAtransfection.

Cytotoxicity Assay

T-cells were incubated in 96-well plates (100,000 cells/well), togetherwith 10,000 target cells (expressing CD123) and 10,000 control(CD123neg) cells in the same well. Target and control cells werelabelled with fluorescent intracellular dyes (CFSE or Cell Trace Violet)before co-culturing them with CAR+ T-cells. The co-cultures wereincubated for 4 hours at 37° C. with 5% CO₂. After this incubationperiod, cells were labelled with a fixable viability dye and analyzed byflow cytometry. Viability of each cellular population (target cells orCD123neg control cells) was determined and the % of specific cell lysiswas calculated. Cytotoxicity assays were carried out 48 h after mRNAtransfection.

T-Cell Transduction

Transduction of T-cells with recombinant lentiviral vectors expressionthe CAR was carried out three days after T-cell purification/activation.CAR detection at the surface of T-cells was done using a recombinantprotein consisting on the fusion of the extracellular domain of thehuman CD123 protein, together with a murine IgG1 Fc fragment. Binding ofthis protein to the CAR molecule was detected with afluorochrome-conjugated secondary antibody targeting the mouse Fcportion of the protein, and analyzed by flow cytometry.

Anti-Tumor Mouse Model

Immunodefficient NOG mice were intravenously (iv) injected with (CD123expressing MOLM13-Luciferase cells as an AML xenograft mouse model.Optionally, mice received an anti-cancer treatment. Mice were then ivinjected (either 2 or 7 days after injection of the tumor cell line)with different doses of CAR+ T-cells to be tested, or with T-cells thatwere not transduced with the CAR lentiviral vector. Bioluminescentsignals were determined at the day of T-cell injection (D0), at D7, 14,21, 28 and 40 after T-cell injection in order to follow tumoralprogression on the different animals.

EXAMPLES Example 1: Proliferation of TCRalpha Inactivated CellsExpressing a CD123-CAR

Heterodimeric TALE-nuclease targeting two 17-bp long sequences (calledhalf targets) separated by an 15-bp spacer within T-cell receptor alphaconstant chain region (TRAC) gene were designed and produced. Each halftarget is recognized by repeats of the half TALE-nucleases listed inTable 10.

TABLE 10 TAL-nucleases targeting TCRalpha gene Target Target sequenceRepeat sequence Half TALE-nuclease TRAC_T01 TTGTCCCACAGATATCCRepeat TRAC_T01-L TRAC_T01-L TALEN Agaaccctgaccctg (SEQ ID NO: 50)(SEQ ID NO: 52) CCGTGTACCAGCTGAGA Repeat TRAC_T01-R TRAC_T01-R TALEN(SEQ ID NO: 49) (SEQ ID NO: 51) (SEQ ID NO: 53)

Each TALE-nuclease construct was subcloned using restriction enzymedigestion in a mammalian expression vector under the control of the T7promoter. mRNA encoding TALE-nuclease cleaving TRAC genomic sequencewere synthesized from plasmid carrying the coding sequence downstreamfrom the T7 promoter.

Purified T cells preactivated during 72 hours with antiCD3/CD28 coatedbeads were transfected with each of the 2 mRNAs encoding both halfTRAC_T01 TALE-nucleases. 48 hours post-transfection, different groups ofT cells from the same donor were respectively transduced with alentiviral vector encoding one of the CD-123 CAR previously described(SEQ ID NO: 23 to 48). 2 days post-transduction, CD3_(NEG) cells werepurified using anti-CD3 magnetic beads and 5 days post-transductioncells were reactivated with soluble anti-CD28 (5 μg/ml).

Cell proliferation was followed for up to 30 days after reactivation bycounting cell 2 times per week. Increased proliferation in TCR alphainactivated cells expressing the CD-123 CARs, especially whenreactivated with anti-CD28, was observed compared to non-transducedcells.

To investigate whether the human T cells expressing the CD123-CARdisplay activated state, the expression of the activation marker CD25are analyzed by FACS 7 days post transduction. The purified cellstransduced with the lentiviral vector encoding CD-123 CAR assayed forCD25 expression at their surface in order to assess their activation incomparison with the non-transduced cells. Increased CD25 expression isexpected both in CD28 reactivation or no reactivation conditions.

Example 2

Construction of CD123 CAR Using Various Anti-CD123 Antibody Fragments

Primary T-Cell Cultures

T cells were purified from Buffy coat samples provided by EFS(Etablissement Francais du Sang, Paris, France) using Ficoll gradientdensity medium (Ficoll Paque PLUS/GE Healthcare Life Sciences). The PBMClayer was recovered and T cells were purified using a commerciallyavailable T-cell enrichment kit (Stem Cell Technologies). Purified Tcells were activated in X-Vivo™-15 medium (Lonza) supplemented with 20ng/mL Human IL-2 (Miltenyi Biotech), 5% Human Serum (Sera Laboratories),and Dynabeads Human T activator CD3/CD28 at a bead:cell ratio 1:1 (LifeTechnologies). After activation cells were grown and maintained inX-Vivo™-15 medium (Lonza) supplemented with 20 ng/mL Human IL-2(Miltenyi Biotech) and 5% Human Serum (Sera Laboratories)

CAR mRNA Transfection

Transfections were done at Day 4 or Day 11 after T-cell purification andactivation. 5 millions of cells were transfected with 15 μg of mRNAencoding the different CAR constructs. CAR mRNAs were produced using themMESSAGE mMACHINE T7 Kit (Life Technologies) and purified using RNeasyMini Spin Columns (Qiagen). Transfections were done using Cytopulsetechnology, by applying two 0.1 mS pulses at 3000V/cm followed by four0.2 mS pulses at 325V/cm in 0.4 cm gap cuvettes in a final volume of 200μl of “Cytoporation buffer T” (BTX Harvard Apparatus). Cells wereimmediately diluted in X-Vivo™-15 media (Lonza) and incubated at 37° C.with 5% CO₂. IL-2 (from Miltenyi Biotech was added 2 h afterelectroporation at 20 ng/mL.

Degranulation Assay (CD107a Mobilization)

T-cells were incubated in 96-well plates (40,000 cells/well), togetherwith an equal amount of cells expressing or not the CD123 protein.Co-cultures were maintained in a final volume of 100 μl of X-Vivo™-15medium (Lonza) for 6 hours at 37° C. with 5% CO₂. CD107a staining wasdone during cell stimulation, by the addition of a fluorescentanti-CD107a antibody (APC conjugated, from Miltenyi Biotec) at thebeginning of the co-culture, together with 1 μg/ml of anti-CD49d (BDPharmingen), 1 μg/ml of anti-CD28 (Miltenyi Biotec), and 1× Monensinsolution (eBioscience). After the 6 h incubation period, cells werestained with a fixable viability dye (eFluor 780, from eBioscience) andfluorochrome-conjugated anti-CD8 (PE conjugated Miltenyi Biotec) andanalyzed by flow cytometry. The degranulation activity was determined asthe % of CD8+/CD107a+ cells, and by determining the mean fluorescenceintensity signal (MFI) for CD107a staining among CD8+ cells.Degranulation assays were carried out 24 h after mRNA transfection.

IFNgamma Release Assay

T-cells were incubated in 96-well plates (40,000 cells/well), togetherwith cell lines expressing or not the CD123 protein. Co-cultures weremaintained in a final volume of 100 μl of X-Vivo™-15 medium (Lonza) for24 hours at 37° C. with 5% CO₂. After this incubation period the plateswere centrifuged at 1500 rpm for 5 minutes and the supernatants wererecovered in a new plate. IFN gamma detection in the cell culturesupernatants was done by ELISA assay (Human IFN-gamma Quantikine ELISAKit, from R&D Systems). The IFN gamma release assays were carried bystarting the cell co-cultures 24 h after mRNA transfection.

Cytotoxicity Assay

T-cells were incubated in 96-well plates (100,000 cells/well), togetherwith 10,000 target cells (expressing CD123) and 10,000 control(CD123neg) cells in the same well. Target and control cells werelabelled with fluorescent intracellular dyes (CFSE or Cell Trace Violet,from Life Technologies) before co-culturing them with CAR+ T-cells. Theco-cultures were incubated for 4 hours at 37° C. with 5% CO₂. After thisincubation period, cells were labelled with a fixable viability dye(eFluor 780, from eBioscience) and analyzed by flow cytometry. Viabilityof each cellular population (target cells or CD123neg control cells) wasdetermined and the % of specific cell lysis was calculated. Cytotoxicityassays were carried out 48 h after mRNA transfection.

Results

6 different scFv's from 7G3, 32716, Klon 43 12F1, 26292, and Old4 wereused to generate Chimeric Antigen Receptors (CARs) and to screen themfor their degranulation activity towards CD123+ cells.

Different architectures were designed (FIG. 2 and FIG. 3) and theiractivity was determined upon transient expression in human T-cells (FIG.5, FIG. 6, FIG. 7 and FIG. 8).

T-cells were purified from buffy-coat samples and activated usingCD3/CD28 beads. 4 days after activation cells were transfected withmRNAs encoding different CAR molecules (using PulseAgileelectroporation) and degranulation activity was assessed 24h aftertransfection.

The results illustrated in FIG. 4 and in FIG. 5 shows degranulationactivity of 6 different scFv's for one single architecture (v3:CD8-hinge/CD8-transmembrane), when CAR+ T-cells were co-cultured for 6hours with CD123 expressing cells (RPMI8226), or with cells that do notexpress CD123 (K562). White bars correspond to degranulation signalsobserved in T-cells that were cultured alone, black bars represent thesignals observed when T-cells were co-cultured with RPMI8226 cells, andgray bars show degranulation signals of T-cells co-cultured with K562cells.

FIG. 4 shows degranulation activity in percentage (%) of degranulationof the 6 different scFv's for one single architecture (v3:CD8-hinge/CD8-transmembrane), when CAR+ T-cells were co-cultured for 6hours with CD123 expressing cells (RPMI8226), or with cells that do notexpress CD123 (K562).

FIG. 5 shows the degranulation activity (CD107a+ cells) in meanfluorescence activity (MFI) of CAR T-cells after 6 h co-cultures withCD123neg cells (K562) or cells expressing high or low levels of CD123(RPMI8226 and KG1a, respectively). The results represent the mean valuesof three independent experiments.

Surprisingly, the results show that although 7G3 is an anti-CD123antibody exhibiting a strong affinity and avidity and in vivoeffectiveness (Jin et al., 2009; Cell Stem Cell 5, 31-42), the CD123 CART cells derived from 7G3 were not active in the present experimentalsettings.

26292-, 32716-, and Klon43-CAR expressing cells exhibited a strongactivity as compared to control-mock) with Klon43 CAR expressing cellsbeing the most active.

Interestingly, whereas 26292 CAR expressing cells and 32716 CARexpressing cells were slightly active towards cells that do not expressCD123 (K562) (grey bars), the activity of Klon43 towards cells that donot express CD123 was comparable to that of Mock T cells.

Among the CAR molecules generated as illustrated in FIG. 2, 8 of themwere selected for further activity tests (FIG. 6, FIG. 7 and FIG. 8).

Construction of CD123 CAR Using Anti-CD123 scFv Antibody FragmentsDerived from Klon43, 12F1, 32716, and 26292 and Functional Analysis

For this, T-cells were isolated from buffy-coat samples and activatedusing CD3/CD28 beads. Cells were transiently transfected with mRNAsencoding the different candidates at D11 after activation. CAR activitywas assessed by measuring their degranulation capacity, the IFN gammarelease, and the cytotoxic activity when co-cultured with cellsexpressing or not CD123.

FIG. 6 shows the degranulation activity (CD107a+ cells) of CAR T-cellsafter 6 h co-cultures with CD123neg cells (K562) or cells expressinghigh or low levels of CD123 (RPMI8226 and KG1a, respectively).Co-cultures were started 24 h after CAR mRNA electroporation. Theresults represent the mean values of three independent experiments.

FIG. 7 shows the amount of IFN gamma released by T-cells whenco-cultured for 24 h with cells expressing different levels of CD123(KG1a or RPMI8226), or with cells that do not express CD123 (K562). IFNgamma release from T-cells cultured alone, in the same conditions thatthe co-cultures, is also shown. The experiments were done for threeindependent donors, and results from a representative donor are shownhere.

FIG. 8 shows the specific cytolytic activity of CAR-T cells. Assays weredone 48 h after CAR mRNA transfection. T-cells were co-cultured withK562+KG1a or K562+RPMI8226 cells for 4 hours. Cellular viability foreach of the cell lines was determined at the end of the co-cultured anda specific cell lysis percentage was calculated.

All constructions were active, with Klon 43 V3 CAR and 32716V3 CARexhibiting the higher activity as compared to control than other CARS.

T-Cell Transduction

Transduction of T-cells with recombinant lentiviral vectors expressionthe CAR was carried out three days after T-cell purification/activation.Lentiviral vectors were produced by Vectalys SA (Toulouse, France) bytransfection of genomic and helper plasmids in HEK-293 cells.Transductions were carried out at a multiplicity of infection of 5,using 10⁶ cells per transduction. CAR detection at the surface ofT-cells was done using a recombinant protein consisting on the fusion ofthe extracellular domain of the human CD123 protein together with amurine IgG1 Fc fragment (produced by LakePharma). Binding of thisprotein to the CAR molecule was detected with a PE-conjugated secondaryantibody (Jackson Immunoresearch) targeting the mouse Fc portion of theprotein, and analyzed by flow cytometry.

Two CAR candidates, namely CAR Klon 43 V3 and CAR 32716V3 were thenselected and cloned into a lentiviral vector, in which CAR expression iscoupled to the BFP through a 2A peptide, and driven by an EF1a promoter.A schematic representation of the lentiviral vector is shown in FIG. 9upper panel. T-cells were isolated from buffy-coat samples, activatedwith CD3/CD28 beads, and transduced 3 days after activation with thelentiviral vectors, at an MOI of 5.

CAR detection was done using a fusion protein in which the extracellulardomain of the human CD123 protein was fused to a mouse IgG1 derived Fcfragment. Binding of the CAR at the cell surface with the CD123 portionof the fusion protein was detected with anti-Fc PE-conjugated antibodyand analyzed by flow cytometry. FIG. 9 represents the % of CAR+ or ofBlue fluorescent protein (BFP+) cells (measured by FACS analysis) at Day8 or 10 post transduction for two different donors.

Activity tests were carried out between D10 and 12 after transduction:

FIG. 10 represents the degranulation activity against different cellslines of transduced cells. Daudi and K562 cells do not express CD123,while KG1a, MOLM13 and RPMI8226 express different levels of CD123(KG1a<MOLM13<RPMI8226). The % of CD107a+ cells (among CD8+ cells) forthree independent donors is given in the upper panel, and the intensityof CD107a staining is shown in the lower panel for a representativedonor. NTD stands for Non Transduced cells.

Again, the data show that the activity (degranulation FIG. 10 or lysisof CD123+ cells FIG. 11) of the anti-CD123 CAR expressing cells derivedfrom Klon 43 V3 is equivalent to that of 32716V3 (FIG. 10 and FIG. 11).

Most surprisingly, 32716V3-derived CAR expressing cells exhibited astronger background activity (activity against cells expressing noCD123, DAUDI and K562) than Klon 43 V3 derived CAR expressing cells(FIG. 10 and FIG. 11).

Klon 43 V3 derived CAR expressing cells had a more specific activity anda slightly but significantly higher activity that, 32716V3-derived CARexpressing cells (FIG. 10, FIG. 11 and FIG. 12) in particular towardcells RPMI8226 cells that express the higher level of CD123. (see FIG.13 and FIG. 14)

FIG. 11 shows the IFN gamma release upon 24 h co-culture of CAR T-cellswith different cell lines.

FIG. 12 shows the shows the specific cytolytic activity of CAR-T cells.T-cells were co-cultured with Daudi+KG1a, Daudi+MOLM13, orDaudi+RPMI8226 cells for 4 hours. Cellular viability for each of thecell lines was determined at the end of the co-cultured and a specificcell lysis percentage was calculated. The results represent resultsobtained in at least two independent donors.

The results indicate that in T-cells stably expressing the CAR,Klon43-v3 displays a slightly higher activity than 32716-v3 in all theactivity tests. In addition, background activity was observed in thedegranulation and IFNgamma release assays, when T-cells expressing the32716-v3 CAR were cultured alone or in the presence of cells that didnot express CD123. This was not observed in T-cells expressing theKlon43-v3 CAR. For these reasons, the Klon43-v3 CAR was selected tocarry out antitumor in vivo experiments in a mouse model.

FIG. 13 and FIG. 14 shows a dose-response degranulation activity foreach of the CAR used. 96-well plates were coated with different doses ofCD123-Fc protein (the same used for detection of CAR+ T-cells, seelegend in FIG. 5), and cells were cultured for 6 h in this plate in thepresence of fluorescent CD107a antibody. The results show thedegranulation activity (% of CD107a+ cells (FIG. 13) and intensity ofCD107a signal (FIG. 14) in CD8+ cells, respectively).

Example 3 Anti-Tumor Mouse Model

Immunodefficient female NOG mice were intravenously (iv) injected withMOLM13-Luciferase cells as an AML xenograft mouse model. NOG(NOD.Cg-Prkdcscidll2rgtm1Sug/JicTac) mice, 6-8 weeks old, were obtainedfrom Taconic (Ry, Danemark) To establish the MOLM13-Luc cell line,MOLM13 cells (DSMZ ACC 554) were transduced with a lentivirus encodingthe GFP and the firefly luciferase (amsbio LVP438-PBS). The GFP-positivecells have been selected with Neomycin (ref 10131-027, Gibco, LifeTechnologies, Saint-Aubin, France). For information, MOLM13 cell linehas been established from the peripheral blood of a 20-year-old man withacute myeloid leukemia AML FAB M5a at relapse in 1995 after initialmyelodysplastic syndromes (MDS, refractory anemia with excess of blasts,RAEB).

Mice were then iv injected (either 2 or 7 days after injection of thetumor cell line) with different doses of CAR+ T-cells (Klon43-v3 CAR),or with T-cells that were not transduced with the CAR lentiviral vector.Bioluminescent signals were determined at the day of T-cell injection(D0) or at D7, 14, 21, 28 and 40 after T-cell injection in order tofollow tumoral progression on the different animals.

Animal housing and experimental procedures were carried out byOncodesign (Dijon, France; http://www.oncodesign.com/), according to theFrench and European Regulations and NRC Guide for the Care and Use ofLaboratory Animals.

Results

FIG. 15 shows the in vivo activity of T-cells expressing the Klon43-v3CAR.

Immunodefficient mice were injected with MOLM13-Luciferase cells either2 or 7 days before injection of non-transduced human T-cells, or withdifferent doses of anti-CD123 CAR+ T-cells. The results represent thebioluminescent signal observed at different time points after T-cellinjection (mean of 4 mice in each group, except for G12, in which 1 ofthe 4 mice died between days 21 and 28).

The data show that the object of the present invention can be usedagainst CD123+ cancer cells, for the treatment of CD123+ cancer humanleukemia cells.

7G3-1 (SEQ ID NO.1 + SEQ ID NO. 23)

LYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 7G3-2 (SEQ ID NO. 1 + SEQ ID NO. 24)

FSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 7G3-3 (SEQ ID NO. 1 + SEQ ID NO. 25)

HTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 7G3-4 (SEQ ID NO. 1 +SEQ ID NO. 26)

HTRGLDFACDIISFFLAITSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 7G3-5 (SEQ ID NO. 1 +SEQ ID NO. 27)

MIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFCSCVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCSRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMGERRRGKGHDGLYQGLSTATKDYDALHMQALPPR 7G3-6 (SEQ ID NO. 1 + SEQ ID NO. 28)

MIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIISFFLALTSTALLFLLFFLTRFSVVKRGRKKLLYIFQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKRRKNPQEGYLNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Old4-3 (SEQ ID NO. 1 + SEQ ID NO. 29)

FACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 26292-1 (SEQ ID NO. 1 +SEQ ID NO. 30)

LAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGCELRVKFRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 26292-2 (SEQ ID NO. 1 + SEQ ID NO. 31)

TSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 26292-3 (SEQ ID NO. 1 + SEQ ID NO. 32)

RPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR26292-4 (SEQ ID NO. 1 + SEQ ID NO. 33)

RPEACRPAAGGAVHTRGLDFACDIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR26292-5 (SEQ ID NO. 1 + SEQ ID NO. 34)

PSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKSKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWQESNGQPENNYKTTPPVLDSDGSFFLYSKLTDKSRWQQGNVSCSVMHEALHNHYTWQKSLSLSPGKIYIWAPLAGTGCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 26292-6 (SEQ ID NO. 1 +SEQ ID NO. 35)

PSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCVMHEALHNHYTQKSLSLSPGKIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 32716-1 (SEQ ID NO. 1 +SEQ ID NO. 36)

YQIYIWAPLAGTCGVLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATDTYDALHMQALPPR 32716-2 (SEQ ID NO. 1 +SEQ ID NO. 37)

YQIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKRRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 32716-3 (SEQ ID NO. 1 +SEQ ID NO. 38)

IASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQLPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP R32716-4 (SEQ ID NO. 1 + SEQ ID NO. 39)

IASQPLSLRPEACRPAAGGAVHTRGLDFACDIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGPRRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATDTYDALHMQALPP R32716-5 (SEQ ID NO. 1 + SEQ ID NO. 40)

PAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVESESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR32716-6 (SEQ ID NO. 1 + SEQ ID NO. 41)

PAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Klo43-1 (SEQ ID NO. 1 +SEQ ID NO. 42)

PEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMGKERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRKlo43-2 (SEQ ID NO. 1 + SEQ ID NO. 43)

FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRKlo43-3 (SEQ ID NO. 1 + SEQ ID NO. 44)

GRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Klo43-4 (SEQ ID NO. 1 + SEQ ID NO. 45)

GRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Klo43-5 (SEQ ID NO. 1 + SEQ ID NO. 46)

HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRKlo43-6 (SEQ ID NO. 1 + SEQ ID NO. 47)

HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR12F1-3 (SEQ ID NO. 1 + SEQ ID NO. 48)

PTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVILLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PR

The invention claimed is:
 1. A method of treating a pre-malignantcancer, a malignant cancer, or a relapse refractory cancer expressingCD123, comprising administering to a patient with a CD123 expressingpre-malignant cancer, malignant cancer or a CD123 expressing relapserefractory cancer a therapeutically effective amount of a pharmaceuticalcomposition comprising an engineered immune cell expressing a CD123specific Chimeric Antigen Receptor (CAR) comprising an extracellularligand binding-domain comprising a heavy chain variable region (V_(H))and a light chain variable region (V_(L)) from a monoclonal anti-CD123antibody, wherein the extracellular ligand domain comprises CDRsequences of SEQ ID NOs: 67, 68, and 69 and SEQ ID NOs: 70, 71, and 72,a FcγRIIIα, CD8α, or IgG1 hinge, a CD8α or 4-1BB transmembrane domain,and a cytoplasmic domain comprising a CD3-ζ signaling domain and aco-stimulatory domain from 4-1BB.
 2. The method of claim 1, wherein theCD123 specific CAR has an amino acid sequence of SEQ ID NO: 42, 44, or46.
 3. The method of claim 1, wherein the malignant cancer condition orthe relapse refractory cancer expressing CD123 is a haematologicalcancer.
 4. The method of claim 3, wherein the haematological cancer is aleukemia or a malignant lymphoproliferative disorder.
 5. The method ofclaim 4, wherein said leukemia is acute myelogenous leukemia (AML),chronic myelogenous leukemia, acute lymphoid leukemia, chronic lymphoidleukemia, or myelodysplastic syndrome.
 6. The method of claim 4, whereinthe leukemia is AML.
 7. The method of claim 4, wherein saidhaematological cancer is a malignant lymphoproliferative disorder. 8.The method of claim 7, wherein said malignant lymphoproliferativedisorder is lymphoma.
 9. The method of claim 8, wherein said lymphoma ismultiple myeloma, non-Hodgkin's lymphoma, Burkitt's lymphoma, small-cellfollicular lymphoma, or large-cell follicular lymphoma.
 10. The methodof claim 1, wherein said V_(H) and V_(L) are humanized.
 11. The methodof claim 1, wherein said V_(H) and V_(L) comprise SEQ ID NO. 73 and/orSEQ ID NO.
 74. 12. The method of claim 1, wherein said V_(H) and V_(L)comprise at least one V_(H) sequence of: SEQ ID Nos: 60-66, and at leastone V_(L) sequence of: SEQ ID Nos: 54-59.
 13. The method of claim 1,wherein said CD123 specific CAR comprises a CD8α hinge and a CD8αtransmembrane domain.
 14. The method of claim 1, wherein said V_(H) andV_(L) comprises an amino acid sequence of SEQ ID NO. 19 or SEQ ID NO.20.
 15. The method of claim 1, wherein said CD123 CAR further comprisesat least one other extracellular ligand binding domain which is notspecific for CD123.
 16. The method of claim 1, wherein said CD123 CARfurther comprises a signal peptide.
 17. The method of claim 1, whereinsaid immune cell is an autologous cell.
 18. The method of claim 1,wherein said immune cell is an allogenic cell.
 19. A method of impairinga hematologic cancer cell expressing CD123 comprising contacting saidhematologic cancer cell with an engineered immune cell expressing aCD123 specific CAR comprising an extracellular ligand binding-domaincomprising V_(H) and V_(L) regions from a monoclonal anti-CD123antibody, wherein the extracellular ligand domain comprises CDRsequences of SEQ ID NOs: 67, 68, and 69 and SEQ ID NOs: 70, 71, and 72,a FcγRIIIα, CD8α, or IgG1 hinge, a CD8α or 4-1BB transmembrane domain,and a cytoplasmic domain comprising a CD3-ζ signaling domain and aco-stimulatory domain from 4-1BB, wherein said engineered immune cellexpressing the CD123 specific CAR is administered in an amount effectiveto cause impairment of said cancer cell.